Inma Castilla-Cortázar

A synthetic peptide from transforming growth factor β type III receptor inhibits liver fibrogenesis in rats with carbon tetrachloride liver injury

Transforming growth factor beta1 (TGF-beta1) is a pleiotropic cytokine, which displays potent profibrogenic effects and is highly expressed in fibrotic livers. For this reason, development of TGF-B1 inhibitors might be of great importance to control liver fibrogenesis as well as other undesired side effects due to this cytokine. Potential peptide inhibitors of TGF-beta1 (derived from TGF-beta1 and from its type III receptor) were tested in vitro and in vivo using different assays. Peptides P11 and P12, derived from TGF-beta1, and P54 and P144, derived from its type III receptor, prevented TGF-beta1-dependent inhibition of MV1Lu proliferation in vitro and markedly reduced binding of TGF-beta1 to its receptors. P144 blocked TGF-beta1-dependent stimulation of a reporter gene under the control of human alpha2(I) collagen promoter. Intraperitoneal administration of P144 also showed potent antifibrogenic activity in vivo in the liver of rats receiving CCl4. These rats also showed a significant decrease in the number of activated hepatic stellate cells as compared with those treated with saline only. These results suggest that short synthetic peptides derived from TGF-beta1 type III receptor may be of value in reducing liver fibrosis in chronic liver injury.

Osteopenia in rats with liver cirrhosis: beneficial effects of IGF-I treatment.

BACKGROUND/AIMS Liver cirrhosis is associated with osteopenia and also with low levels of IGF-I. This hormone has been reported to stimulate bone formation in states of undernutrition and low bone turnover. Our aims were to evaluate whether osteopenia develops in male Wistar rats with CCl4-induced cirrhosis and whether IGF-I is effective in the restoration of bone mass in these animals. METHODS Cirrhotic rats were distributed into two groups: group CI (n = 12) which received placebo and group CI + IGF (n = 12) which was treated with human recombinant IGF-I (2 microg/100 g bw/day, s.c., 21 days). Twelve normal animals which received placebo constituted the control group. On the 22nd day, the animals were sacrificed, and bone parameters were analyzed in femur and/or tibia. RESULTS Posterior-anterior and latero-medial diameters were similar in all groups. Also, no significant differences were observed in bone contents of calcium, total proteins, collagen and hydroxyapatite in CI rats as compared with controls. However, CI rats showed significant reductions in bone weight (-13.5%, p < 0.001), total bone density (-9.28%, p < 0.001), and increased perimedullar bone resorption and urinary levels of deoxypyridinoline (a marker of bone resorption). In CI + IGF rats these parameters improved significantly as compared with CI animals. CONCLUSIONS Osteopenia characterized by loss of bone mass and preserved bone composition is found in rats with CCl4-induced cirrhosis. This bone disorder is partially corrected by treatment with low doses of IGF-I. Since osteoporosis seems to be the predominant form of osteopenia in patients with cirrhosis, IGF-I should be considered as a possible therapy for this disorder.

Low doses of insulin-like growth factor-I improve nitrogen retention and food efficiency in rats with early cirrhosis

BACKGROUND/AIMS In order to ascertain whether malnutrition is an early-onset feature of liver cirrhosis and whether the anabolic hormone insulin-like growth factor I (IGF-I) could be useful in the treatment of this complication, we analyzed the nutritional alterations present in rats with early-stage liver cirrhosis and the effects of IGF-I on nutritional parameters in these animals. METHODS After a 24 h fast, a 15N-enriched diet was administered for 5 days to normal control rats and to cirrhotic rats receiving subcutaneous injections of vehicle (Group 1) or IGF-I, 2 micrograms.100 g bw-1.day-1, (Group 2) during the 5 experimental days. 15N, a stable N isotope, was measured in biological samples by mass spectrometry. RESULTS Compared with control rats, Group 1 animals showed significant reductions in N intake and food efficiency (p < 0.05, both). In addition, the weight of the gastrocnemius muscle, its total N content and the dietary N content of this muscle were significantly lower in Group 1 than in control animals (p < 0.05, all). In rats from Group 2, mean values of N intake, food efficiency, gastrocnemius N content and the amount of dietary N incorporated into this muscle were similar to those in control rats, and (with the exception of gastrocnemius N total content) significantly higher than those in non-treated cirrhotic rats (p < 0.05, all). CONCLUSIONS A variety of nutritional disturbances were detected in rats from the early stages of liver cirrhosis. Low doses of IGF-I were found to reverse most of these changes. These results stimulate further studies to determine whether IGF-I might be useful in the correction of the malnutrition present in patients with liver cirrhosis.

Antifibrogenic effect in vivo of low doses of insulin-like growth factor-I in cirrhotic rats.

Insulin-like growth factor-I (IGF-I) is produced mainly in the liver and it induces beneficial effects on the nutritional status, the liver function and oxidative hepatic damage in cirrhotic rats. The aim of this work was to analyze the effect of IGF-I on mechanisms of fibrogenesis in cirrhotic rats. Liver cirrhosis was induced by CCl(4) inhalation and phenobarbital in Wistar rats. Ten days after stopping CCl(4) administration (day 0), rats received either IGF-I (2 microg/100 g bw/day) (CI+IGF) or saline (CI) subcutaneously during 14 days. Animals were sacrificed on day 15. As control groups were used: healthy rats (CO) and healthy rats treated with IGF-I (CO+IGF). Liver histopathology, hydroxyproline content, prolyl hydroxylase activity, collagen I and III mRNA expression and the evolution of transformed Ito cells into myofibroblasts were assessed. Among the two control groups (CO+IGF), no differences were found in hydroxyproline content and these levels were lower than those found in the two cirrhotic groups. Compared with untreated cirrhotic rats, the CI+IGF-I animals showed a significant reduction in hydroxyproline content, prolyl hydroxylase activity and collagen alpha 1(I) and alpha1(III) mRNA expression. A higher number of transformed Ito cells (alpha-actin +) was observed in untreated cirrhotic animals as compared to CO and CI+IGF groups. In summary, treatment with IGF-I reduced all of the studied parameters of fibrogenesis. In conclusion, low doses of IGF-I induce in vivo an antifibrogenic effect in cirrhotic rats.

Jejunal microvilli atrophy and reduced nutrient transport in rats with advanced liver cirrhosis: improvement by Insulin-like Growth Factor I

BackgroundPrevious results have shown that in rats with non-ascitic cirrhosis there is an altered transport of sugars and amino acids associated with elongated microvilli. These alterations returned to normal with the administration of Insulin-Like Growth Factor-I (IGF-I). The aims of this study were to explore the evolution of these alterations and analyse the effect of IGF-I in rats with advanced cirrhosis and ascites. Thus, jejunal structure and nutrient transport (D-galactose, L-leucine, L-proline, L-glutamic acid and L-cystine) were studied in rats with ascitic cirrhosis.MethodsAdvanced cirrhosis was induced by CCl4 inhalation and Phenobarbital administration for 30 weeks. Cirrhotic animals were divided into two groups which received IGF-I or saline during two weeks. Control group was studied in parallel. Jejunal microvilli were studied by electron microscopy. Nutrient transport was assessed in brush border membrane vesicles using 14C or 35S-labelled subtracts in the three experimental groups.ResultsIntestinal active Na+-dependent transport was significantly reduced in untreated cirrhotic rats. Kinetic studies showed a decreased Vmax and a reduced affinity for sugar and four amino acids transporters (expressed as an increased Kt) in the brush border membrane vesicles from untreated cirrhotic rats as compared with controls. Both parameters were normalised in the IGF-I-treated cirrhotic group. Electron microscopy showed elongation and fusion of microvilli with degenerative membrane lesions and/or notable atrophy.ConclusionsThe initial microvilli elongation reported in non ascitic cirrhosis develops into atrophy in rats with advanced cirrhosis and nutrient transports (monosaccharides and amino acids) are progressively reduced. Both morphological and functional alterations improved significantly with low doses of IGF-I.

Effects of IGF-I treatment on osteopenia in rats with advanced liver cirrhosis.

IGF-I is an anabolic hormone which has been reported to increase bone formation in several conditions of undernutrition. Advanced liver cirrhosis is associated with osteopenia and also with low serum levels of IGF-I. Previous results showed that low doses of IGF-I increase osteoblastic activity and decrease bone reabsoption in early liver cirrhosis. The aim of this study was to evaluate whether IGF-I-treatment also induces beneficial effect on osteopenia associated with advanced cirrhosis. Rats with ascitic cirrhosis were divided into two groups: group CI (n=10) which received saline and group CI+IGF (n=10) which were treated with IGF-I (2 μg/100 g bw. day, sc, during 21 days). Healthy controls which received saline were studied in parallel (CO n=10). On the 22nd day, the animals were sacrificed, and bone parameters were analyzed in femur. Posterior-anterior diameter was similar in all groups. No significant differences were observed in bone content of calcium, total proteins, collagen and hydroxyapatite in cirrhotic rats as compared with controls. However, CI rats showed significant reductions in total bone density (−13.5%, p<0.001) assessed by densitometry) and radiological study. In CI+IGF rat bone density (assesed by densitometry) improved significantly as compared with CI animals. In summary, osteopenia characterized by loss of bone mass and preserved bone composition was found in rats with advanced cirrhosis induced by CCl4 and phenobarbital in drinking water. This bone disorder is partially restored by treatment with low doses of IGF-I during only three weeks. Thus, IGF-I could be considered as a possible therapy for osteopenia associated with advanced liver cirrhosis.ResumenIGF-I es una hormona anabólica con efecto sobre el metabolismo óseo. La cirrosis hepática avanzada está asociada con osteopenia y también con bajas concentraciones de IGF-I en suero. Resultados anteriores de nuestro grupo muestran que dosis bajas de IGF-I aumentan la actividad osteoblástica y reducen la resorción ósea en estadíos compensados de cirrosis hepática. El objetivo de este estudio consiste en evaluar si el tratamiento con IGF-I induce también un efecto beneficioso en la osteopenia asociada a cirrosis avanzada. Ratas con cirrosis ascítica se dividen en dos grupos: grupo CI (n=10), que reciben salino, y grupo CI+IGF (n=10) tratadas con IGF-I (2mg/100 g peso corporal/día, sc, durante 21 días). Controles sanos, que reciben salino, se estudian al mismo tiempo (CO n=10). El día 22, los animales se sacrifican y se analizan parámetros óseos en fémur. El diámetro antero-posterior es similar en todos los grupos. No se observan tampoco diferencias significativas en el contenido óseo (calcio, proteínas totales, colágeno, hidroxiapatita) en las ratas cirróticas al compararlas con los controles. Sin embargo, en los animales cirróticos se observa una reducción significativa de la densidad total ósea (−13.5%, p<0.001) medida por densitometría, reducción que se aprecia también en el estudio radiológico. En las ratas CI+IGF la densidad ósea mejora significativamente en comparación con los animales cirróticos sin tratamiento. En resumen, se observa osteopenia caracterizada por pérdida de masa ósea, en la que se preserva la normal composición del hueso, en ratas con cirrosis avanzada inducida por CCl4 y fenobarbital en el agua de bebida. Esta disminución de la masa ósea se recupera parcialmente por el tratamiento con dosis bajas de IGF-I durante sólo tres semanas. Por lo tanto, IGF-I podre ser considerado como una posible terapia para la osteopenia asociada a la cirrosis hepática en estadíos avanzados.

IGF1 gene transfer into skeletal muscle using recombinant adeno-associated virus in a rat model of liver cirrhosis

Systemic administration of recombinant IGF1 at low levels has been shown to improve hepatic function, nutritional status and testicular atrophy in rats with CCl4-induced cirrhosis. We have developed a recombinant adeno-associated (rAAV) viral vector containing the cDNA for rat IGF1 and confirmed the expression of IGF1 after intramuscular injection of this vector in a rat model of liver cirrhosis. Although weight of injected muscles was significantly increased in rats with mild cirrhosis, this was not the case in rats with advanced, de-compensated cirrhosis. Furthermore, we found no significant amelioration of liver damage in treated rats at any stage of liver cirrhosis. Our results suggest that IGF1 gene transfer into muscle results in a local effect, at least at the vector dose employed here.ResumenLa administración sistémica de IGF1 a bajos niveles provoca mejoría de la función hepática, el estado nutricional y la atrofia testicular de las ratas con cirrosis hepática inducida por CCl4. En este trabajo, se ha desarrollado un vector viral adeno-asociado (rAAV) que contiene el cDNA de IGF1 de rata y se ha confirmado la expresión de IGF1 tras la inyección intramuscular de dicho vector en un modelo de cirrosis hepática en rata. Aunque el peso de los músculos tratados aumenta significativamente en ratas con cirrosis incipiente, este efecto no se reproduce en ratas con cirrosis avanzada y descompensada. Por otra parte, no se puede objetivar una mejoría del daño hepático en las ratas tratadas, independientemente del estado de la cirrosis que presentan. Nuestros resultados sugieren que la transferencia génica de IGF1 al músculo provoca un efecto local, al menos a las dosis de vector empleadas en este estudio.

IGF-I does not improve fat malabsorption in cirrhotic rats.

The aim of this study was to explore whether IGF-I treatment for two weeks modified fat intestinal absorption, measured from faecal content, in cirrothic and healthy rats. Male Wistar rats were used. Liver cirrhosis was induced by carbon tetrachloride (CCl4) inhalation twice a week for 11 weeks and phenobarbital addition to drinking water (400 mg/l) 1 week before an during the period of CCl4 exposure. Both control and cirrhotic rats were randomly assigned to two different groups to receive .the vehicle or IGF-I (2 mg/100mg bw day). The results show that faecal lipid content in cirrhotic animals significantly increases as compaired to control groups and no differences are found between untreated and IGF-treated cirrhotic animals. In summary, fat malabsorption observed in cirrothic animals is not corrected by the treatment with IGF-I