Hepatic remodeling, serum biomarkers and prevention of fibrosis progression in liver disease.

Abstract

Hepatic fibrosis is characterized by intense tissue remodeling. In this study, we assessed whether CO3-610, a new identified neoepitope, could be used as a surrogate biomarker of liver fibrosis and portal hypertension in CCl4-induced experimental fibrosis. Serum CO3-610 was measured by ELISA. Liver fibrosis was quantified by Sirius red staining. Serum hyaluronic acid (HA) was measured with a binding-protein assay. Gene expression of collagens I and III, MMP2 and MMP9, and tissue inhibitors of matrix metalloproteinase 1 and 2 was quantified by PCR. Hemodynamic measurements were taken in a subgroup of animals. A close direct relationship was found between serum CO3-610 and hepatic collagen content (r= 0.78; P<0.001), superior to that found for serum HA (r= 0.49; P<0.05). CO3-610 levels in rats with severe fibrosis (43.5±3.3 ng/mL, P<0.001) and cirrhosis (60.6±4.3 ng/mL, P<0.001) were significantly higher than those in control animals (26.6±1.3 ng/mL). Importantly, a highly significant relationship was found between serum CO3-610 and portal hypertension (r= 0.84; P<0.001). Liver MMP9 expression increased significantly in fibrotic animals but decreased to control levels in cirrhotic ones. Circulating CO3-610 behaves as a reliable indicator of hepatic remodeling and portal hypertension in experimental fibrosis. Therefore, this peptide could ultimately be a useful marker for the management of liver disease in patients.

Endocannabinoids behave as antifibrogenic agents by interacting with cannabinoid CB2 receptors, whereas the apelin (AP) system acts as a proangiogenic and profibrogenic mediator in the liver. This study assessed the effect of long-term stimulation of CB2 receptors or AP receptor (APJ) blockade on fibrosis progression in rats under a non-discontinued fibrosis induction program. The study was performed in control and CCl4-treated rats for 13 weeks. Fibrosis-induced rats received a CB2 receptor agonist (AM1241) (1 mg/kg), an APJ antagonist (F13A) (75 μg/kg), or vehicle daily during the last 5 weeks of the CCl4 inhalation program. Mean arterial pressure (MAP), portal pressure (PP), hepatic collagen content, angiogenesis, cell infiltrate, and mRNA expression of a panel of fibrosis-related genes were measured in all animals. Fibrosis-induced rats showed increased hepatic collagen content, reduced MAP, PP, and increased expression of the assessed messengers in comparison with control rats. However, fibrotic rats treated with either AM1241 or F13A had reduced hepatic collagen content, improved MAP and PP, ameliorated cell viability, and reduced angiogenesis and cell infiltrate compared with untreated fibrotic rats. These results were associated with attenuated induction of PDGFRβ, α-SMA, MMPs, and TIMPs. CB2 receptor stimulation or APJ blockade prevents fibrosis progression in CCl4-treated rats. The mechanisms underlying these phenomena are coincident despite the marked dissimilarities between the CB2 and APJ signaling pathways, thus opening new avenues for preventing fibrosis progression in liver diseases. PDGF is the most potent stimulus for proliferation and migration of stellate cells. PDGF receptor β expression is an important phenotypic change in myofibroblastic cells that mediates proliferation and chemotaxis. Here we analyzed the relationship between PDGFRβ expression, hemodynamic deterioration, and fibrosis. Thereafter, we investigated the effects produced by an adenovirus encoding a dominant-negative soluble PDGFRβ (sPDGFRβ) on hemodynamic parameters, PDGFRβ signaling pathway, and fibrosis. Hemodynamics, PDGFRβ mRNA expression, and hepatic collagen were assessed in controls and fibrosic/cirrhotic rats. Next, 30 fibrotic rats were randomized into three groups receiving iv saline and an adenovirus encoding for sPDGFRβ or β-galactosidase. After 7days, hemodynamics, serum sPDGFRβ, and hepatic collagen were measured. CCl4-treated animals for 18weeks showed a significantly higher increase in PDGFRβ mRNA compared to those treated for 13weeks and control rats. In CCl4-treated rats, the fibrous tissue area ranged from moderate to severe fibrosis. A direct relationship between the degree of fibrosis, hemodynamic changes, and PDGFRβ expression was observed. Fibrotic rats transduced with the adenovirus encoding sPDGFRβ showed increased MAP, decreased PP, lower activation of the PDGFRβ signaling pathway, and reduced hepatic collagen than fibrotic rats receiving β-gal or saline. PDGFRβ activation closely correlates with hemodynamic disorders and increased fibrosis in CCl4-treated rats. Adenoviral dominant negative soluble PDGFRβ improved fibrosis. As a result, the hemodynamic abnormalities were ameliorated.

Cirrhotic patients have altered host-defense response mechanisms. Here we assessed whether impaired expression of CB2 receptor in monocytic cells of cirrhotic patients could be involved in the pathogenesis of this phenomenon. CB2 mRNA and protein expression was assessed in a differentiated human monocytic cell line (U937) stimulated with endotoxin (LPS). A PCR array of 86 different genes was assessed in U937 cells treated with LPS. A migration assay towards endocannabinoids or the CB2 antagonist, SR144528, was performed in U937 cells exposed to LPS. Finally, CB1 and CB2 mRNA expression were measured in monocytes and macrophages of cirrhotic patients with or without spontaneous bacterial peritonitis. LPS reduced CB2 expression in human monocytes. Endocannabinoids increased the migratory activity of U937 cells, which was reverted when the experiments were performed in the presence of LPS. Transcriptional profiling showed marked upregulation of 9 genes related to proinflammatory signaling. However, only two genes encoding for CB1 and CB2 were reduced in LPS-treated cells. Circulating monocytes of cirrhotic patients showed a significantly diminished mRNA expression of CB1 and CB2. Markedly low CB1 and CB2 mRNA levels were found in peritoneal macrophages of cirrhotic patients with ascites, being almost suppressed when analyzed in patients with peritonitis. LPS reduces CB2 expression in human monocytes resulting in depressed chemotactic activity and therefore impaired host defense response of these cells.

La característica más destacable de la fibrosis es la desregulación de la ME. El equilibrio que existe entre la síntesis y la degradación de la ME en un hígado normal se pierde, y como consecuencia se favorece la síntesis y acumulación de fibras de colágeno, el cual provoca la distorsión de la arquitectura del parénquima y de la red vascular hepática. El CO3-610 es un producto de degradación del colágeno III que ha mostrado estar correlacionado significativamente con el aumento de la cantidad de colágeno y progresión de la fibrosis. Los valores máximos de CO3-610 se han encontrado ratas cirróticas. El resultado más importante es que el CO3-610 ha mostrado tener una estrecha correlación con los valores de presión portal. Existe un gran interés en investigar y desarrollar terapias dirigidas a la prevención de la progresión de la fibrosis. Los endocannabinoides son moléculas lipídicas que participan en un amplio rango de procesos fisiológicos. Los efectos son mediados por dos tipos de receptores, el CB1 y el CB2. El sistema de la apelina es otro sistema endógeno que ha despertado mucho interés en los últimos años. La apelina es el único ligando conocido para el receptor APJ. El tratamiento con AM1241, un agonista selectivo de CB2 y F13A, un antagonista de APJ mejoró la función hepática, la hemodinámica sistémica y portal, y provocó una reducción significativa del grado de fibrosis hepática. Ambos tratamientos produjeron una disminución del infiltrado inflamatorio, angiogénesis y del grado de apoptosis en el parénquima hepático. El análisis de la expresión del mRNA de varios genes importantes implicados en el proceso fibrogénico, reveló que el tratamiento tanto con AM1241 como con F13A, producían una disminución de la expresión del PDGFRβ, del αSMA y de genes de remodelado tisular. La inhibición parcial de la vía de PDGF mediante la administración de un dominante negativo para la fracción soluble del receptor β de PDGF, ha demostrado que mejora la hemodinámica sistémica y la presión portal además de mejorar el grado de fibrosis. Por último, los pacientes cirróticos con ascitis tienen una mayor probabilidad de sufrir infecciones (peritonitis bacteriana espontánea, PBE). Se ha observado que estos pacientes tienen una menor expresión de CB2. In vitro se han realizado experimentos con células monocíticas (U937) y se ha observado que el LPS disminuye drásticamente la expresión de CB2 al igual que afecta a la capacidad de migración de estas células. En conjunto, se puede concluir que la evaluación de la fibrosis hepática es una herramienta muy importante para el diagnostico de la enfermedad hepática y que éste puede mejorarse a través del uso de biomarcadores no invasivos que correlacionen con la hemodinámica y el grado de fibrosis hepática. La prevención de la progresión de la fibrosis a través de fármacos o bien actuando sobre genes clave involucrados en la fibrogénesis son posibles dianas terapéuticas. Finalmente, es posible mejorar la calidad de vida de los pacientes cirróticos con ascitis con infección por PBE mediante terapias que involucren los receptores de cannabinoides CB2.