An adenosine derivative promotes mitochondrial supercomplexes reorganization and restoration of mitochondria structure and bioenergetics in a diethylnitrosamine-induced hepatocellular carcinoma model.

Hepatocellular carcinoma (HCC) progression is associated with dysfunctional mitochondria and bioenergetics impairment. However, no data about the relationship between mitochondrial supercomplexes (hmwSC) formation and ATP production rates in HCC are available. Our group has developed an adenosine derivative, IFC-305, which improves mitochondrial function, and it has been proposed as a therapeutic candidate for HCC. We aimed to determine the role of IFC-305 on both mitochondrial structure and bioenergetics in a sequential cirrhosis-HCC model in rats. Our results showed that IFC-305 administration decreased the number and size of liver tumors, reduced the expression of tumoral markers, and reestablished the typical architecture of the hepatic parenchyma. The livers of treated rats showed a reduction of mitochondria number, recovery of the mtDNA/nDNA ratio, and mitochondrial length. Also, IFC-305 increased cardiolipin and phosphatidylcholine levels and promoted hmwSC reorganization with changes in the expression levels of hmwSC assembly-related genes. IFC-305 in HCC modified the expression of several genes encoding elements of electron transport chain complexes and increased the ATP levels by recovering the complex I, III, and V activity. We propose that IFC-305 restores the mitochondrial bioenergetics in HCC by normalizing the quantity, morphology, and function of mitochondria, possibly as part of its hepatic restorative effect.

Liver fibrotic development is reduced through inflammation prevention by an adenosine derivative compound.

BACKGROUND: Liver fibrosis is a global health problem, and studying its development provides important information to address its treatment. Here, we characterized the effects of an adenosine compound (IFC-305) on preventing fibrosis and liver inflammation. METHODS: We studied the impact of IFC-305 on a carbon tetrachloride-induced liver fibrosis model in Wistar male rats at 4, 6, and 8 weeks. The effects were characterized by liver tissue histology, macrophages identification by flow cytometry with CD163+/CD11b/c+ antibodies, hepatic and plasmatic cytokine levels employing MILLIPLEX MAP and ELISA, Col1a1 and Il6 gene expression by RTqPCR, lipoperoxidation by TBARS reaction, and reactive oxygen species using 2'-7'dichlorofluorescin diacetate. RESULTS: CCl4-induced liver fibrosis and inflammation were significantly reduced in rats treated with IFC-305 at 6 and 8 weeks. In addition, we observed diminished expression of Col1a1; a decrease in the inflammatory cytokines IL-1ß, IL-6, MCP-1, TNF-α, and IL-4 a; reduction in inflammatory macrophages; inhibition of lipoperoxidation; and ROS production in Kupffer cells. CONCLUSION: This study showed that IFC-305 can inhibit liver fibrosis establishment by regulating the immune response during CCl4-induced damage. The immunomodulatory action of IFC-305 supports its use as a potential therapeutic strategy for preventing liver fibrosis.

Expression and distribution of dopamine transporter in cardiac tissues of the guinea pig.

Dopamine transporter (DAT) is a membrane protein that it is a marker for dopaminergic neurons. In the present work, throught Western blot and autoradiographic studies with a selective ligand for DAT ([(3)H] WIN-35428) and noradrenaline transporter (NET) ([(3)H] Nisoxetine), we search the expression and distribution of DAT in comparison with NET, in cardiac tissue of guinea pig in order to support the presence of dopaminergic nerve cells into the heart. Expression of DAT, and NET were evidenced by a bands of 75 and 54 kDa, respectively in the heart. Binding for DAT and NET were found in the four cardiac chambers. However, DAT show heterogeneous distribution with binding in right atria and in both ventricles, whereas NET show homogenous distribution in the four cardiac chambers. The results show the expression of DAT in cardiac tissues with a different distribution compared with NET, being an evidence for the presence of dopaminergic nerve cells into the heart.

Prevention of in vitro hepatic stellate cells activation by the adenosine derivative compound IFC305.

We have previously shown that adenosine and the aspartate salt of adenosine (IFC305) reverse pre-established CCl(4)-induced cirrhosis in rats. However, their molecular mechanism of action is not clearly understood. Hepatic stellate cells (HSC) play a pivotal role in liver fibrogenesis leading to cirrhosis, mainly through their activation, changing from a quiescent adipogenic state to a proliferative myofibrogenic condition. Therefore, we decided to investigate the effect of IFC305 on primary cultured rat HSC. Our results reveal that this compound suppressed the activation of HSC, as demonstrated by the maintenance of a quiescent cell morphology, including lipid droplets content, inhibition of α-smooth muscle actin (α-SMA) and collagen α1(I) expression, and up-regulation of MMP-13, Smad7, and PPARγ expression, three key antifibrogenic genes. Furthermore, IFC305 was able to repress the platelet-derived growth factor (PDGF)-induced proliferation of HSC. This inhibition was independent of adenosine receptors stimulation; instead, IFC305 was incorporated into cells by adenosine transporters and converted to AMP by adenosine kinase. On the other hand, addition of pyrimidine ribonucleoside as uridine reversed the suppressive effect of IFC305 on the proliferation and activation of HSC, suggesting that intracellular pyrimidine starvation would be involved in the molecular mechanism of action of IFC305. In conclusion, IFC305 inhibits HSC activation and maintains their quiescence in vitro; these results could explain in part the antifibrotic liver beneficial effect previously described for this compound on the animal model.

An adenosine derivative compound, IFC305, reverses fibrosis and alters gene expression in a pre-established CCl(4)-induced rat cirrhosis.

Cirrhosis is a complex process that involves a dynamic modification of liver cell phenotype associated to gene expression changes. This study investigates the reversing capacity of an adenosine derivative compound (IFC305) on a rat model of liver cirrhosis and gene expression changes associated with it. Rats were treated with IFC305 or saline for 5 or 10 weeks after cirrhosis induction (CCl(4) treatment for 10 weeks). Fibrosis score, collagenase activity and amount of hepatic stellate cells (HSC, activated and with a lipid-storing phenotype) were measured in livers. In addition, gene expression analysis was performed using 5K DNA microarrays and quantitative RT-PCR. Treatment of cirrhotic rats with IFC305 for 5 or 10 weeks compared to saline control, induced: (1) reduction of fibrosis (50-70%) and of collagen, of alpha-SMA and desmin proteins, as well as of activated HSCs in liver, (2) increased collagenase activity and cell number of lipid-storing HSC, (3) improved serum parameters of liver function, such as reduced activity of aminotransferases and bilirubin. Expression of 413 differential genes, deregulated in cirrhotic samples, tended to be normalized by IFC305 treatment. Some genes modulated at transcript level by IFC305 were Tgfb1, Fn1, Col1a1, C9, Apoa1, Ass1, Cps1, and Pparg. The present study shows that IFC305 reverses liver fibrosis through modulation of adipogenic and fibrosis-related genes and by ameliorating hepatic function. Thus, understanding of the anti-cirrhotic effect of IFC305 might have therapeutical potential in patients with cirrhosis.

Correlation between oxidative stress and alteration of intracellular calcium handling in isoproterenol-induced myocardial infarction.

Myocardial Ca(2+) overload and oxidative stress are well documented effects associated to isoproterenol (ISO)-induced myocardial necrosis, but information correlating these two issues is scarce. Using an ISO-induced myocardial infarction model, 3 stages of myocardial damage were defined: pre-infarction (0-12 h), infarction (12-24 h) and post-infarction (24-96 h). Alterations in Ca(2+) homeostasis and oxidative stress were studied in mitochondria, sarcoplasmic reticulum and plasmalemma by measuring the Ca(2+) content, the activity of Ca(2+) handling proteins, and by quantifying TBARs, nitric oxide (NO) and oxidative protein damage (changes in carbonyl and thiol groups). Free radicals generated system, antioxidant enzymes and oxidative stress (GSH/GSSG ratio) were also monitored at different times of ISO-induced cardiotoxicity. The Ca(2+) overload induced by ISO was counterbalanced by a diminution in the ryanodine receptor activity and the Na(+)-Ca(+2) exchanger as well as by the increase in both calcium ATPases activities (vanadate- and thapsigargine-sensitive) and mitochondrial Ca(2+) uptake during pre-infarction and infarction stages. Pro-oxidative reactions and antioxidant defences during the 3 stages of cardiotoxicity were observed, with maximal oxidative stress during the infarction. Significant correlations were found among pro-oxidative reactions with plasmalemma and sarcoplasmic reticulum Ca(2+) ATPases, and ryanodine receptor activities at the onset and development of ISO-induced infarction. These findings could be helpful in the design of antioxidant therapies in this pathology.