María García-Fernández

Oxidative stress in fibroblasts from patients with pseudoxanthoma elasticum: possible role in the pathogenesis of clinical manifestations†

Pseudoxanthoma elasticum (PXE) is a genetic disease characterized by calcification and fragmentation of elastic fibres of the skin, cardiovascular system and eye, caused by mutations of the ABCC6 gene, which encodes the membrane transporter MRP6. The pathogenesis of the lesions is unknown. Based on studies of similar clinical and histopathological damage present in haemolytic disorders, our working hypothesis is that PXE lesions may result from chronic oxidative stress occurring in PXE cells as a consequence of MRP6 deficiency. Our results show that PXE fibroblasts suffer from mild chronic oxidative stress due to the imbalance between production and degradation of oxidant species. The findings also show that this imbalance results, at least in part, from the loss of mitochondrial membrane potential (ΔΨm) with overproduction of H2O2. Whether mitochondrial dysfunction is the main factor responsible for the oxidative stress in PXE cells remains to be elucidated. However, mild chronic generalized oxidative stress could explain the great majority of structural and biochemical alterations already reported in PXE. Copyright

Low Doses of Insulin-Like Growth Factor-I Induce Mitochondrial Protection in Aging Rats

Serum IGF-I levels decline with age. We have recently reported that in aging rats the exogenous administration of IGF-I restores IGF-I circulating levels and age related-changes, improving glucose and lipid metabolisms, increasing testosterone levels and serum total antioxidant capability, and reducing oxidative damage in the brain and liver associated with a normalization of antioxidant enzyme activities. Understanding that mitochondria are one of the most important cellular targets of IGF-I, the aims of this study were to characterize mitochondrial dysfunction and study the effect of IGF-I therapy on mitochondria, leading to cellular protection in the following experimental groups: young controls, untreated old rats, and aging rats treated with IGF-I. Compared with young controls, untreated aging rats showed an increase of oxidative damage in isolated mitochondria with a mitochondrial dysfunction characterized by: depletion of membrane potential with increased proton leak rates and intramitochondrial free radical production, and a significant reduction of ATPase and complex IV activities. In addition, mitochondrial respiration from untreated aging rats was atractyloside insensitive, suggesting that the adenine nucleotide translocator was uncoupled. The adenine nucleotide translocator has been shown to be one of the most sensitive locations for pore opening. Accordingly, untreated aging rats showed a significant overexpression of the active fragment of caspases 3 and 9. IGF-I therapy corrected these parameters of mitochondrial dysfunction and reduced caspase activation. In conclusion, these results show that the cytoprotective effect of IGF-I is closely related to a mitochondrial protection, leading to reduce free radical production, oxidative damage, and apoptosis, and to increased ATP production.

Low Doses of Insulin-Like Growth Factor I Improve Insulin Resistance, Lipid Metabolism, and Oxidative Damage in Aging Rats

GH and IGF-I concentrations decline with age. Age-related changes appear to be linked to decreases in the anabolic hormones, GH and IGF-I. The aim of this study was to investigate the antioxidant, anabolic, and metabolic effects of the IGF-I replacement therapy, at low doses, in aging rats. Three experimental groups were included in this protocol: young healthy controls (17 wk old); untreated old (O) rats (103 wk old); and aging rats (103 wk old) treated with IGF-I during 1 month (2.25 microg IGF-I/100 g body weight(-1).d(-1)). Compared with young controls, untreated aging rats showed a reduction of IGF-I and testosterone levels, and a decrease of serum total antioxidant status, which were corrected by IGF-I therapy. In addition, untreated O presented increased levels of serum glucose with hyperinsulinemia, cholesterol, and triglycerides, and a reduction of free fatty acid concentrations. IGF-I therapy was able to revert insulin resistance, and to reduce cholesterol and triglycerides levels increasing significantly free fatty acid concentrations. The O group showed higher oxidative damage in brain and liver tissues associated with alterations in antioxidant enzyme activities. IGF-I therapy reduced oxidative damage in brain and liver, normalizing antioxidant enzyme activities and mitochondrial dysfunction. In conclusion, low doses of IGF-I restore circulating IGF-I, improve glucose and lipid metabolism, increase testosterone levels and serum total antioxidant capability, and reduce oxidative damage in brain and liver associated with a normalization of antioxidant enzyme activities and mitochondrial function.

Parameters of oxidative stress are present in the circulation of PXE patients

Pseudoxanthoma elasticum (PXE) is an inherited disorder characterized by calcification of elastic fibres leading to dermatological and vascular alterations associated to premature aged features and to life threatening clinical manifestations. The severity of the disease is independent from the type of mutation in the ABCC6 gene, and it has been suggested that local and/or systemic factors may contribute to the occurrence of clinical phenotype. The redox balance in the circulation of 27 PXE patients and of 50 healthy subjects of comparable age was evaluated by measuring the advanced oxidation protein products (AOPP), the lipid peroxidation derivatives (LOOH), the circulating total antioxidant status (TAS), the thiol content and the extracellular superoxide dismutase activity (EC-SOD). Patients were diagnosed by clinical, ultrastructural and molecular findings. Compared to control subjects, PXE patients exhibited significantly lower antioxidant potential, namely circulating TAS and free thiol groups, and higher levels of parameters of oxidative damage, as LOOH and of AOPP, and of circulating EC-SOD activity. Interestingly, the ratio between oxidant and antioxidant parameters was significantly altered in PXE patients and related to various score indices. This study demonstrates, for the first time, that several parameters of oxidative stress are modified in the blood of PXE patients and that the redox balance is significantly altered compared to control subjects of comparable age. Therefore, in PXE patients the circulating impaired redox balance may contribute to the occurrence of several clinical manifestations in PXE patients, and/or to the severity of disease, thus opening new perspectives for their management.

Fibroblast protein profile analysis highlights the role of oxidative stress and vitamin K recycling in the pathogenesis of pseudoxanthoma elasticum

Pseudoxanthoma elasticum (PXE) is a genetic disorder associated to mutations in the ABCC6 gene; however, the pathogenetic mechanisms leading to elastic fibre calcifications and to clinical manifestations are still unknown. Dermal fibroblasts, directly involved in the production of the extracellular milieu, have been isolated from healthy subjects and from patients affected by PXE, cultured in vitro and characterized for their ability to produce reactive oxygen species, for structural and functional properties of their cell membranes, for changes in their protein profile. Data demonstrate that oxidative stress has profound and endurable consequences on PXE fibroblast phenotype being responsible for: reduced levels of global DNA methylation, increased amount of carbonylated proteins and of lipid peroxidation products, altered structural properties of cell membranes, modified protein expression. Data shed new light on the pathogenetic pathways in PXE, by identifying a network of proteins affecting elastic fibre calcification through inefficient vitamin K recycling, and highlight the role of differentially expressed proteins as targets for validating the efficacy of future therapeutic strategies aiming to delay and/or revert the pathologic phenotype of PXE fibroblasts. Moreover, data open new perspectives for investigating PXE‐like phenotypes in the absence of ABCC6 mutations.

Hepatoprotection and neuroprotection induced by low doses of IGF-II in aging rats

BackgroundGH and IGFs serum levels decline with age. Age-related changes appear to be associated to decreases in these anabolic hormones. We have previously demonstrated that IGF-I replacement therapy improves insulin resistance, lipid metabolism and reduces oxidative damage (in brain and liver) in aging rats. Using the same experimental model, the aim of this work was to study whether the exogenous administration of IGF-II, at low doses, acts analogous to IGF-I in aging rats.MethodsThree experimental groups were included in this study: young healthy controls (yCO, 17 weeks old); untreated old rats (O, 103 weeks old); and aging rats treated with IGF-II (O+IGF-II, 2 μg * 100 g body weight-1 * day-1) for 30 days. Analytical parameters were determined in serum by routine laboratory methods using an autoanalyzer (Cobas Mira; Roche Diagnostic System, Basel, Switzerland). Serum levels of hormones (testosterone, IGF-I and insulin) were assessed by RIA. Serum Total Antioxidant Status was evaluated using a colorimetric assay. Mitochondrial membrane potential was evaluated using rhodamine 123 dye (adding different substrates to determine the different states). ATP synthesis in isolated mitochondria was determined by an enzymatic method.ResultsCompared with young controls, untreated old rats showed a reduction of IGF-I and testosterone levels with a decrease of serum total antioxidant status (TAS). IGF-II therapy improved serum antioxidant capability without modifying testosterone and IGF-I circulating concentrations. In addition, IGF-II treatment reduced oxidative damage in brain and liver, improving antioxidant enzyme activities and mitochondrial function. IGF-II was also able to reduce cholesterol and triglycerides levels increasing free fatty acids concentrations.ConclusionsWe demonstrate that low doses of IGF-II induce hepatoprotective, neuroprotective and metabolic effects, improving mitochondrial function, without affecting testosterone and IGF-I levels.

Fear extinction and acute stress reactivity reveal a role of LPA(1) receptor in regulating emotional-like behaviors.

LPA1 receptor is one of the six characterized G protein-coupled receptors (LPA1–6) through which lysophosphatidic acid acts as an intercellular signaling molecule. It has been proposed that this receptor has a role in controlling anxiety-like behaviors and in the detrimental consequences of stress. Here, we sought to establish the involvement of the LPA1 receptor in emotional regulation. To this end, we examined fear extinction in LPA1-null mice, wild-type and LPA1 antagonist-treated animals. In LPA1-null mice we also characterized the morphology and GABAergic properties of the amygdala and the medial prefrontal cortex. Furthermore, the expression of c-Fos protein in the amygdala and the medial prefrontal cortex, and the corticosterone response following acute stress were examined in both genotypes. Our data indicated that the absence of the LPA1 receptor significantly inhibited fear extinction. Treatment of wild-type mice with the LPA1 antagonist Ki16425 mimicked the behavioral phenotype of LPA1-null mice, revealing that the LPA1 receptor was involved in extinction. Immunohistochemistry studies revealed a reduction in the number of neurons, GABA+ cells, calcium-binding proteins and the volume of the amygdala in LPA1-null mice. Following acute stress, LPA1-null mice showed increased corticosterone and c-Fos expression in the amygdala. In conclusion, LPA1 receptor is involved in emotional behaviors and in the anatomical integrity of the corticolimbic circuit, the deregulation of which may be a susceptibility factor for anxiety disorders and a potential therapeutic target for the treatment of these diseases.

Liver mitochondrial dysfunction is reverted by insulin-like growth factor II (IGF-II) in aging rats

BackgroundSerum IGF-I and IGF-II levels decline with age. IGF-I replacement therapy reduces the impact of age in rats. We have recently reported that IGF-II is able to act, in part, as an analogous of IGF-I in aging rats reducing oxidative damage in brain and liver associated with a normalization of antioxidant enzyme activities. Since mitochondria seem to be the most important cellular target of IGF-I, the aim of this work was to investigate whether the cytoprotective actions of IGF-II therapy are mediated by mitochondrial protection.MethodsThree groups of rats were included in the experimental protocol young controls (17 weeks old); untreated old rats (103 weeks old); and aging rats (103 weeks old) treated with IGF-II (2 μg/100 g body weight and day) for 30 days.ResultsCompared with young controls, untreated old rats showed an increase of oxidative damage in isolated mitochondria with a dysfunction characterized by: reduction of mitochondrial membrane potential (MMP) and ATP synthesis and increase of intramitochondrial free radicals production and proton leak rates. In addition, in untreated old rats mitochondrial respiration was not blocked by atractyloside. In accordance, old rats showed an overexpression of the active fragment of caspases 3 and 9 in liver homogenates. IGF-II therapy corrected all of these parameters of mitochondrial dysfunction and reduced activation of caspases.ConclusionsThe cytoprotective effects of IGF-II are related to mitochondrial protection leading to increased ATP production reducing free radical generation, oxidative damage and apoptosis.

Effect of IGF-I on total serum antioxidant status in cirrhotic rats.

Articulo en colaboracion con: Maria Fernandez Garcia, Matias Diaz Sanchez, Fernando Diez Caballero, Alberto Castilla, A. Diaz Casares, I. Varela Nieto, Santiago Gonalez Baron

Ectopic calcification in β-thalassemia patients is associated with increased oxidative stress and lower MGP carboxylation.

A number of beta-thalassemia (β-thal) patients in the course of the disease exhibit ectopic calcification affecting skin, eyes and the cardiovascular system. Clinical and histopathological features have been described similar to those in pseudoxanthoma elasticum (PXE), although different genes are affected in the two diseases. Cultured dermal fibroblasts from β-thal patients with and without PXE-like clinical manifestations have been compared for parameters of redox balance and for the expression of proteins, which have been already associated with the pathologic mineralisation of soft connective tissues. Even though oxidative stress is a well-known condition of β-thal patients, our results indicate that the occurrence of mineralized elastin is associated with a more pronounced redox disequilibrium, as demonstrated by the intracellular increase of anion superoxide and of oxidized proteins and lipids. Moreover, fibroblasts from β-thal PXE-like patients are characterized by decreased availability of carboxylated matrix Gla protein (MGP), as well as by altered expression of proteins involved in the vitamin K-dependent carboxylation process. Results demonstrate that elastic fibre calcification is promoted when redox balance threshold levels are exceeded and the vitamin K-dependent carboxylation process is affected decreasing the activity of MGP, a well-known inhibitor of ectopic calcification. Furthermore, independently from the primary gene defect, these pathways are similarly involved in fibroblasts from PXE and from β-thal PXE-like patients as well as in other diseases leading to ectopic calcification, thus suggesting that can be used as markers of pathologic mineralisation.