Publications (20)65.84 Total impact
- [Show abstract] [Hide abstract] ABSTRACT: Sepsis and septic shock are the major causes of death in intensive care units. Oxidative damage to mitochondria is involved in the development of organ dysfunction associated with sepsis. This syndrome is caused by an excessive defensive and inflammatory response characterised by a massive increases of reactive oxygen species (ROS), nitric oxide (NO) and inflammatory cytokines. Under normal circumstances, complex interacting antioxidant defense systems control oxidative stress within mitochondria The consequences of sepsis is a systemic damage to the vascular endothelium, impaired tissue and a compromised whole body respiration, antioxidant depletion and mitochondrial respiratory dysfunction with diminished levels of ATP and O2 consumption. In general, ROS are essential to the functions of cells and particularly immune cells, but adequate levels of antioxidant defenses are required to protect against the harmful effects of excessive ROS production. This review considers the process of sepsis from a mitochondrial perspective, discussing strategies for the targeted delivery of antioxidants to mitochondria. We will provide a summary of the following areas: the cellular metabolism of ROS and its role in pathophysiological processes such as sepsis; currently available antioxidants and possible reasons for their efficacy and inefficacy in ameliorating oxidative stress-mediated diseases; and recent developments in mitochondria-targeted antioxidants and the future implications for such approaches in patients.
- [Show abstract] [Hide abstract] ABSTRACT: The medical use of nitroglycerin (GTN) is limited by patient tolerance. The present study evaluated the role of mitochondrial Complex I in GTN biotransformation and the therapeutic effect of mitochondrial antioxidants. The development of GTN tolerance (in rat and human vessels) produced a decrease in mitochondrial O(2) consumption. Co-incubation with the mitochondria-targeted antioxidant mitoquinone (MQ, 10(-6)mol/L) or with glutathione ester (GEE, 10(-4)mol/L) blocked GTN tolerance and the effects of GTN on mitochondrial respiration and aldehyde dehydrogenase 2 (ALDH-2) activity. Biotransformation of GTN depended on the mitochondria being functionally active, particularly mitochondrial Complex I. Tolerance induced mitochondrial ROS production and oxidative stress, though these effects were not detected in HUVECρ(0) cells or Complex I mutant cells. Experiments performed to evaluate Complex I-dependent respiration demonstrated that its inhibition by GTN was prevented by the antioxidants in control samples. These results point to a key role for mitochondrial Complex I in the adequate functioning of ALDH-2. In addition, we have identified mitochondrial Complex I as one of the targets at which the initial oxidative stress responsible for GTN tolerance takes place. Our data also suggest a role for mitochondrial-antioxidants as therapeutic tools in the control of the tolerance that accompanies chronic nitrate use.
- [Show abstract] [Hide abstract] ABSTRACT: The current interest in developing Glycine transporter Type 1 (GlyT-1) inhibitors, for diseases such as schizophrenia, has led to the demand for a GlyT-1 PET molecular imaging tool to aid drug development and dose selection. We report on [(11) C]GSK931145 as a novel GlyT-1 imaging probe in primate and man. Primate PET studies were performed to determine the level of specific binding following homologous competition with GSK931145 and the plasma-occupancy relationship of the GlyT-1 inhibitor GSK1018921. Human PET studies were performed to determine the test-retest reproducibility of [(11) C]GSK931145 and the plasma-occupancy relationship of GSK1018921. [(11) C]GSK931145 entered primate and human brain and yielded a heterogeneous pattern of uptake which was similar in both species with highest uptake in midbrain, thalamus, and cerebellum. Homologous competition in primates indicated no viable reference region and gave binding potential estimates between 1.5 and 3 for midbrain, thalamus and cerebellum, While the distribution and binding potential values were similar across species, both the plasma free fraction (f(P) : 0.8 vs. 8%) and delivery (K(1) : 0.025 vs. 0.126 ml cm(-3) min(-1) ) were significantly lower in humans. Test-retest reproducibility in humans calculated using a two tissue compartmental model was poor (VAR(V(T) ): 29-38%), but was improved using a pseudo reference tissue model (VAR(BP(ND) ): 16-23%). GSK1018921 EC(50) estimates were 22.5 and 45.7 ng/ml in primates and humans, respectively.
- [Show abstract] [Hide abstract] ABSTRACT: Whereas molecular imaging studies in the aging human brain have predominantly demonstrated reductions in serotonin transporter (5-HTT) availability, the majority of the rodent studies, using autoradiographic methods, report increases in neural 5-HTT levels with age. To our knowledge, however, no previous rodent studies have assessed this topic in vivo, and therefore it remains unclear whether this discrepancy arises from methodological or inter-species differences. We performed an [(11)C]-DASB microPET study to evaluate the effects of aging on 5-HTT availability in the rat brain. To generate binding potential estimates, quantitative tracer kinetic modeling was applied using the simplified reference tissue model. A global increase in whole-brain [(11)C]-DASB binding potential was observed in the aged rats in comparison to the control group. More specifically, regional analyses revealed a highly significant increase in 5-HTT binding in the medial frontal cortex, and more modest increments in the midbrain/thalamus. Our results suggest that the frontal cortex represents a site of robust age-related alterations in the rat serotonergic system, and stress the need for further research assessing this topic in the human frontal cortex. Moreover, these findings suggest that the reported discrepancies between rodent and human data may reflect a divergence in the aging processes affecting human and rat serotonergic terminals.
- [Show abstract] [Hide abstract] ABSTRACT: Diabetes is a chronic disease and as a consequence of the overproduction of reactive oxygen species (ROS), is related with oxidative stress. There are different sources of ROS, of which mitochondria is the main one. Oxidative stress seems to play an important role in mitochondria- mediated disease processes, though the exact molecular mechanisms responsible remain elusive. There are evidences which supports the idea that impaired mitochondrial function is a cause of the insulin insensitivity in different type of cells that arised as a result of an insufficient supply of energy or defects in the insulin signaling pathway. ROS are generally necessary for the proper functioning of the cell, but excessive ROS production can be harmful, which makes antioxidant defenses essential. Moreover, some substances with antioxidant properties, such as vitamin C or vitamin E, erradicate the oxidative stress associated with diabetes. The results of clinical trials employing anti-oxidative stress reagents in patients with diabetes are contradictory, which may be a result of inadequate study design or selected targets. This review considers the process of diabetes from a mitochondrial perspective, and describes the role of autophagy in the development of diabetes. Furthermore, we discuss the possible beneficial effects of selectively targeting antioxidants to mitochondria as a strategy for modulating mitochondrial function in diabetes.
- [Show abstract] [Hide abstract] ABSTRACT: In this study, we assessed the feasibility of using positron emission tomography (PET) and the tracer [¹¹C]OMAR ([¹¹C]JHU75528), an analogue of rimonabant, to study the brain cannabinoid type 1 (CB1) receptor system. Wild-type (WT) and CB1 knockout (KO) animals were imaged at baseline and after pretreatment with blocking doses of rimonabant. Brain uptake in WT animals was higher (50%) than in KO animals in baseline conditions. After pretreatment with rimonabant, WT uptake lowered to the level of KO animals. The results of this study support the feasibility of using PET with the radiotracer [¹¹C]JHU75528 to image the brain CB1 receptor system in mice. In addition, this methodology can be used to assess the effect of new drugs in preclinical studies using genetically manipulated animals.
Dataset: Figure S1[Show abstract] [Hide abstract] ABSTRACT: Analysis of TK activity by 18F-FEAU substrate. (A) NP-18 pancreatic cancer cells were transduced at the indicated viral doses. Twenty-four hours later cells were incubated with 0,91 µCi of 18F-FEAU for 2 h and radioactivity was quantified in the cell extracts. (B) Tumors from CWR-CLT cells stably expressing the TK gene were s.c. injected into the flanks of nude mice.18F-FEAU tumor retention was analyzed as described in materials and methods. Images show specific signal in the tumor. (C) Mice bearing BxPC-3 xenografts received three intratumoral injections of 2×1010 vp/tumor AdTK. Five days later 18F-FEAU retention was analyzed. Images show TK activity in the tumor and strong signal in the liver. (TIF)
- [Show abstract] [Hide abstract] ABSTRACT: Replication-competent adenoviruses armed with thymidine kinase (TK) combine the concepts of virotherapy and suicide gene therapy. Moreover TK-activity can be detected by noninvasive positron emission-computed tomography (PET) imaging, what could potentially facilitate virus monitoring in vivo. Here, we report the generation of a novel oncolytic adenovirus that incorporates the Tat8-TK gene under the control of the Major Late Promoter in a highly selective backbone thus providing selectivity by targeting the retinoblastoma pathway. The selective oncolytic TK virus, termed ICOVIR5-TK-L, showed reduced potency compared to a non-selective counterpart. However the combination of ICOVIR5-TK-L with ganciclovir (GCV) induced a potent antitumoural effect similar to that of wild type adenovirus in a preclinical model of pancreatic cancer. Although the treatment with GCV provoked a reduction in the viral yield, both in vitro and in vivo, a two-cycle treatment of virus and GCV resulted in an enhanced antitumoral response that correlated with high TK-activity, based on microPET measurements. Thus, TK-expressing oncolytic adenoviruses can be traced by PET imaging providing real time information on the activity of the virus and its antitumoral potency can be optimized by GCV dosing.
Dataset: Figure S2[Show abstract] [Hide abstract] ABSTRACT: Virus release and TK expression. HEK 293 cells were seeded at 2×105 cells per well in triplicate in 24-well plates and cultured overnight. The next day, cells were infected with ICOVIR5-TK-L at 1×103 vp per cell. Four hours later, infection medium was removed and cells were washed three-times with PBS and incubated with fresh medium. (A) At the indicated time points a fraction of the supernatant was harvested and viral yield was determined by the anti-hexon staining method. Detection of viral particles became evident at 48 h post-infection. (B) RT-PCR analysis was performed to determine TK and E1A expression in RNA extracted from cell pellets at the indicated time-points. E1A and TK were first detected at 6 h and 24 h post-infection respectively. (TIF)
- [Show abstract] [Hide abstract] ABSTRACT: (11)C-GSK931145 is a novel radioligand suitable for imaging the glycine transporter 1 (GlyT-1) in brain. In the present study, human dosimetry is estimated from baboon and human biodistribution data. Three baboons and eight healthy human volunteers underwent whole-body positron emission tomography (PET) scans. Human dosimetry was estimated using three different region-of-interest (ROI) delineation methods that ranged in their complexity and execution time: ROIs drawn on anterior-posterior compressed PET images, on subsamples of the organs, and covering the whole-organ. Residence times for each organ were calculated as the area under the time-activity curves divided by the injected activity. Radiation dose estimates were calculated from organ residence times using the OLINDA/EXM software package. The overall distribution of activity was similar in baboons and humans. Early scans presented high activity in the liver, and moderate activity in the lungs and kidneys. The principal route of clearance was intestinal and no urinary excretion was observed. The limiting organ with the highest radiation-absorbed dose was the liver. The mean effective dose in humans was 4.02 μSv/MBq (male phantom) and 4.95 μSv/MBq (female phantom) (ROIs drawn on subsamples of the organs). The human effective dose estimated from baboon data was ~15% larger than the effective dose estimated from human data. Human PET imaging of the glycine transporter-1 with (11)C-GSK931145 results in a moderate effective human radiation dose, which allows for multiple PET examinations in the same individual. Among the three methods compared to delineate ROIs, the organ subsampling method shows the best balance between quantitative accuracy and practical application.
- [Show abstract] [Hide abstract] ABSTRACT: Mitochondrial dysfunction plays a key role in sepsis. We used a sepsis model of human endothelial cells (HUVEC) to study mitochondrial function during normoxic (21% O(2)) and hypoxic (1% O(2)) conditions. When stimulated with a LPS cocktail, HUVEC displayed an increase of nitric oxide (NO) in normoxic and hipoxic conditions, being higher at 21% O(2). LPS-activation for 24 h at 1% O(2) increased ROS production, which was reversed with the mitochondrial antioxidant Mitoquinone (MQ) and Glutathione Ethyl Ester (GEE). Activated cells displayed diminished mitochondrial O(2) consumption with specific inhibition of Complex I, accompanied by increase in tyrosine nitration and Type II NOS protein expression, effects which were recovered by antioxidants and/or with L-NAME. These parameters varied with O(2) environment, namely inhibition of respiration observed in both O(2) environments at 24 h was very similar, whereas O(2) consumption rate fell earlier in 1% O(2)-exposed cells. While no significant differences were detected at earlier time points, at 24 h tyrosine nitration was higher in normoxic vs. hypoxic cells. Mitochondria are heavily implicated in sepsis. Mitochondrial antioxidants provide a mechanistic model for the development of potential therapies.
- [Show abstract] [Hide abstract] ABSTRACT: This study evaluates the performance of simultaneous dual-tracer technique (SDTT) in static positron emission tomography (PET) studies using 2-deoxy-2-[¹⁸F]fluoro-D-glucose and [¹³N]ammonium as radiotracers. The effects of applying SDTT either to the reconstructed image or directly to the sinogram, different rebinning algorithms, total acquisition time, and frame duration were investigated; first, using a specific phantom and later using an in vivo application of the study of cerebral ischemia. The best results were obtained using the image method with single-slice rebinning and a total acquisition time of at least 20 min. Frame duration did not affect SDTT performance. The method was also applied in rats with transient cerebral ischemia to simultaneously study cerebral blood flow and cerebral glucose metabolism. The results encourage the use of SDTT as it has very good potential for examining two different biological processes at the same time utilising rodent PET scanners.
- [Show abstract] [Hide abstract] ABSTRACT: The GABA-ergic system, known to regulate neural tissue genesis during cortical development, has been postulated to play a role in cerebral aging processes. Using in vivo molecular imaging and voxel-wise quantification, we aimed to assess the effects of aging on the benzodiazepine (BDZ) recognition site of the GABA(A) receptor. To visualize BDZ site availability, [(11)C]-flumazenil microPET acquisitions were conducted in young and old rats. The data were analyzed and region of interest analyses were applied to validate the voxel-wise approach. We observed decreased [(11)C]-flumazenil binding in the aged rat brains in comparison with the young control group. More specifically, clusters of reduced radioligand uptake were detected in the bilateral hippocampus, cerebellum, midbrain, and bilateral frontal and parieto-occipital cortex. Our results support the pertinence of voxel-wise quantification in the analysis of microPET data. Moreover, these findings indicate that the aging process involves declines in neural BDZ recognition site availability, proposed to reflect alterations in GABA(A) receptor subunit polypeptide expression.
- [Show abstract] [Hide abstract] ABSTRACT: Functional impairment of endothelial activity (endothelial dysfunction) precedes the development of cardiovascular diseases (CVD). This condition is a result of a reduced bioavailability of nitric oxide (NO), a well known vasodilator, which is mainly due to increased NO degradation caused by its reaction with reactive oxygen species (ROS). Although there are several conditions that contribute independently to endothelial dysfunction, such as hyperglycemia, insulin resistance, hyperinsulinemia and dyslipidemia, increased oxidative stress seems to play a key role. In addition to their original pharmacological properties, drugs used clinically at present, including anti-hypertension reagents, angiotensin receptor blockers and anti-hyperlipidemic reagents such as statins, protect various organs via anti-oxidative stress mechanisms. Moreover, some substances with antioxidant properties, such as vitamin C or vitamin E, have been used to eradicate the oxidative stress associated with CVD. The results of the clinical trials employing anti-oxidative stress reagents in patients with CVD are contradictory, which could be a result of inadequate study design or selected targets. This review considers the process of endothelial dysfunction and CVD from a mitochondrial perspective and evaluates strategies currently under development for the targeted delivery of antioxidants or NO to mitochondria. It endorses the idea that selectively targeting specific antioxidants and NO donors to mitochondria is an effective strategy for modulating mitochondrial respiration and ROS production and protecting mitochondria against oxidative stress.
- [Show abstract] [Hide abstract] ABSTRACT: Rodent models are frequently used in aging research to investigate biochemical age effects and aid in the development of therapies for pathological and non-pathological age-related degenerative processes. In order to validate the use of animal models in aging research and pave the way for longitudinal intervention-based animal studies, the consistency of cerebral aging processes across species needs to be evaluated. The dopaminergic system seems particularly susceptible to the aging process, and one of the most consistent findings in human brain aging research is a decline in striatal D2-like receptor (D2R) availability, quantifiable by positron emission tomography (PET) imaging. In this study, we aimed to assess whether similar age effects can be discerned in rat brains, using in vivo molecular imaging with the radioactive compound [(11)C]-raclopride. We observed a robust decline in striatal [(11)C]-raclopride uptake in the aged rats in comparison to the young control group, comprising a 41% decrement in striatal binding potential. In accordance with human studies, these results indicate that substantial reductions in D2R availability can be measured in the aged striatal complex. Our findings suggest that rat and human brains exhibit similar biochemical alterations with age in the striatal dopaminergic system, providing support for the pertinence of rodent models in aging research.
- [Show abstract] [Hide abstract] ABSTRACT: [(11)C]NNC112 (8-chloro-7-hydroxy-3-methyl-5-(7-benzofuranyl)-2,3,4,5-tetrahydro-IH-3-benzazepine), a selective positron-emission tomography (PET) ligand for the D(1) receptor (R) over the 5-HT(2A) R in vitro, has shown lower selectivity in vivo, hampering measurement of D(1) R in the cortex. [(11)C]NNC112 PET and intravenous (i.v) ketanserin challenge were used to (1) confirm the previous findings of [(11)C]NNC112 in vivo D(1) R selectivity, and (2) develop a feasible methodology for imaging cortical D(1) R without contamination by 5-HT(2A) R. Seven healthy volunteers underwent [(11)C]NNC112 PET scans at baseline and after a 5-HT(2A) R-blocking dose of ketanserin (0.15 mg/kg, i.v.). Percent BP(ND) change between the post-ketanserin and baseline scans was calculated. Irrespective of the quantification method used, ketanserin pretreatment led to significant decrease of BP(ND) in the cortical (approximately 30%) and limbic regions (approximately 20%) but not in the striatum, which contains a much lower amount of 5-HT(2A) R. Therefore, ketanserin allows D(1) R signal to be detected by [(11)C]NNC112 PET without significant 5-HT(2A) R contamination. These data confirm the presence of a significant 5-HT(2A) R contribution to cortical [(11)C]NNC112 signal, and call for caution in the interpretation of published [(11)C]NNC112 PET findings on cortical D(1) R in humans. In the absence of more selective ligands, [(11)C]NNC112 PET with ketanserin can be used for cortical D(1) R imaging in vivo.
- [Show abstract] [Hide abstract] ABSTRACT: Chronic and acute overproduction of reactive oxygen species (ROS) under pathophysiologic conditions forms an integral part of the development of cardiovascular diseases (CVD), and in particular atherosclerosis. These ROS are released from different sources, such as xanthine oxidase, lipoxygenase, nicotinamide adenine dinucleotide phosphate oxidase, the uncoupling of nitric oxide synthase and, in particular, mitochondria. Endothelial dysfunction, characterized by a loss of nitric oxide (NO) bioactivity, occurs early on in the development of atherosclerosis, and determines future vascular complications. Although the molecular mechanisms responsible for mitochondria-mediated disease processes are not clear, oxidative stress seems to play an important role. In general, ROS are essential to cell function, but adequate levels of antioxidant defenses are required in order to avoid the harmful effects of excessive ROS production. Mitochondrial oxidative stress damage and dysfunction contribute to a number of cell pathologies that manifest themselves through a range of conditions. This review considers the process of atherosclerosis from a mitochondrial perspective, and assesses strategies for the targeted delivery of antioxidants to mitochondria that are currently under development. We will provide a summary of the following areas: the cellular metabolism of reactive oxygen species (ROS) and its role in pathophysiological processes such as atherosclerosis; currently available antioxidants and possible reasons for their efficacy and inefficacy in ameliorating oxidative stress-mediated diseases; and recent developments in mitochondrially-targeted antioxidants that concentrate on the matrix-facing surface of the inner mitochondrial membrane in order to protect against mitochondrial oxidative damage, and their therapeutic potential as a treatment for atherosclerosis.
Parc de recerca biomedica de barcelonaBarcino, Catalonia, Spain
Institut Marqués, Spain, BarcelonaBarcino, Catalonia, Spain