Antonio J. López-Farré

Role of Mitochondrial Dysfunction in Hypertension and Obesity

Mitochondria are essential for the maintenance of normal physiological function of tissue cells. Mitochondria are subject to dynamic processes in order to establish a control system related to survival or cell death and adaptation to changes in the metabolic environment of cells. Mitochondrial dynamics includes fusion and fission processes, biogenesis, and mitophagy. Modifications of mitochondrial dynamics in organs involved in energy metabolism such as the pancreas, liver, skeletal muscle, and white adipose tissue could be of relevance for the development of insulin resistance, obesity, and type 2 diabetes. Mitochondrial dynamics and the factors involved in its regulation are also critical for neuronal development, survival, and function. Modifications in mitochondrial dynamics in either agouti-related peptide (AgRP) or pro-opiomelanocortin (POMC), circuits which regulates feeding behavior, are related to changes of food intake, energy balance, and obesity development. Activation of the sympathetic nervous system has been considered as a crucial point in the pathogenesis of hypertension among obese individuals and it also plays a key role in cardiac remodeling. Hypertension-related cardiac hypertrophy is associated with changes in metabolic substrate utilization, dysfunction of the electron transport chain, and ATP synthesis. Alterations in both mitochondrial dynamics and ROS production have been associated with endothelial dysfunction, development of hypertension, and cardiac hypertrophy. Finally, it might be postulated that alterations of mitochondrial dynamics in white adipose tissue could contribute to the development and maintenance of hypertension in obesity situations through leptin overproduction. Leptin, together with insulin, will induce activation of sympathetic nervous system with consequences at renal, vascular, and cardiac levels, driving to sodium retention, hypertension, and left ventricular hypertrophy. Moreover, both leptin and insulin will induce mitochondrial alterations into arcuate nucleus leading to signals driving to increased food intake and reduced energy expenditure. This, in turn would perpetuate white adipose tissue excess and its well-known metabolic and cardiovascular consequences.

Identification of a new locus and validation of previously reported loci showing differential methylation associated with smoking. The REGICOR study

Smoking increases the risk of many diseases and could act through changes in DNA methylation patterns. The aims of this study were to determine the association between smoking and DNA methylation throughout the genome at cytosine-phosphate-guanine (CpG) site level and genomic regions. A discovery cross-sectional epigenome-wide association study nested in the follow-up of the REGICOR cohort was designed and included 645 individuals. Blood DNA methylation was assessed using the Illumina HumanMethylation450 BeadChip. Smoking status was self-reported using a standardized questionnaire. We identified 66 differentially methylated CpG sites associated with smoking, located in 38 genes. In most of these CpG sites, we observed a trend among those quitting smoking to recover methylation levels typical of never smokers. A CpG site located in a novel smoking-associated gene (cg06394460 in LNX2) was hypomethylated in current smokers. Moreover, we validated two previously reported CpG sites (cg05886626 in THBS1, and cg24838345 in MTSS1) for their potential relation to atherosclerosis and cancer diseases, using several different approaches: CpG site methylation, gene expression, and plasma protein level determinations. Smoking was also associated with higher THBS1 gene expression but with lower levels of thrombospondin-1 in plasma. Finally, we identified differential methylation regions in 13 genes and in four non-coding RNAs. In summary, this study replicated previous findings and identified and validated a new CpG site located in LNX2 associated with smoking.

New circulating biomarkers for predicting cardiovascular death in healthy population.

There is interest to analyse newer biomarkers to identify healthy individuals at risk to develop cardiovascular disease (CVD) incidents and death. To determine in healthy individuals new circulating protein biomarkers, whose systemic levels may be associated with the risk of future development of CVD incidents and death. The study was performed in 82 individuals from the Malmö Diet and Cancer study cohort, free from CVD of whom 41 developed CVD and 41 did not. Plasma proteins related to inflammation and thrombo‐coagulating processes were analysed. α1‐antitrypsin isotype 3 plasma levels were significantly higher while apolipoprotein J plasma levels were lower in participants that developed CVD incidents than those that did not develop acute cardiovascular episode. Of 82 participants, 17 died by CVD causes. There were proteins whose expression in plasma was significantly higher in participants suffering CVD death as compared with those that did not die by CVD. These proteins included: fibrinogen β‐chain isotypes 1 and 3, fibrinogen‐γ‐chain isotype 2, vitamin D‐binding protein isotypes 1, 2 and 3, α1‐antitrypsin isotypes 3 and 6, haptoglobin isotypes 3,4,5 and 5, haemopexin isotypes 1 and 2, and Rho/Rac guanine nucleotide exchange factor 2. Moreover, apolipoprotein J plasma levels were found lower in participants that died by cardiovascular cause. Association between plasma levels of proteins and CVD death was independent of age, gender, conventional risk factors and plasma C‐reactive protein levels. Several protein plasma levels and protein isotypes related to inflammation and thrombo‐coagulating phenomena were independently associated with the risk of future CVD death.

Nitric oxide from mononuclear cells may be involved in platelet responsiveness to aspirin

Several mechanisms have been proposed to explain why some platelets have a reduced response to aspirin (ASA). Among them, it was reported an increased circulating level of vitamin‐D‐binding protein (DBP). In addition, nitric oxide (NO) released from mononuclear cells was involved in the antiplatelet effects of ASA. The aim was to analyse the relationship between platelet response to ASA and both NO generation and vitamin‐D‐binding protein content in mononuclear cells.

Platelet Content of Nitric Oxide Synthase 3 Phosphorylated At Serine1177 Is Associated with the Functional Response of Platelets to Aspirin

Objective To analyse if platelet responsiveness to aspirin (ASA) may be associated with a different ability of platelets to generate nitric oxide (NO). Patients/Methods Platelets were obtained from 50 patients with stable coronary ischemia and were divided into ASA-sensitive (n = 26) and ASA-resistant (n = 24) using a platelet functionality test (PFA-100). Results ASA-sensitive platelets tended to release more NO (determined as nitrite + nitrate) than ASA-resistant platelets but it did not reach statistical significance. Protein expression of nitric oxide synthase 3 (NOS3) was higher in ASA-sensitive than in ASA-resistant platelets but there were no differences in the platelet expression of nitric oxide synthase 2 (NOS2) isoform. The highest NOS3 expression in ASA-sensitive platelets was independent of the presence of T-to-C mutation at nucleotide position −786 (T−786→C) in the NOS3-coding gene. However, platelet content of phosphorylated NOS3 at Serine (Ser)1177, an active form of NOS3, was higher in ASA-sensitive than in ASA-resistant platelets. The level of platelet NOS3 Ser1177 phosphorylation was positively associated with the closure time in the PFA-100 test. In vitro, collagen failed to stimulate the aggregation of ASA-sensitive platelets, determined by lumiaggregometry, and it was associated with a significant increase (p = 0.018) of NOS3 phosphorylation at Ser1177. On the contrary, collagen stimulated the aggregation of ASA-resistant platelets but did not significantly modify the platelet content of phosphorylated NOS3 Ser1177. During collagen stimulation the release of NO from ASA-sensitive platelets was significantly enhanced but it was not modified in ASA-resistant platelets. Conclusions Functional platelet responsiveness to ASA was associated with the platelet content of phosphorylated NOS3 at Ser1177.

Metabolic differences between white and brown fat from fasting rabbits at physiological temperature

It has been suggested that activated brown adipose tissue (BAT) shows increased glucose metabolic activity. However, less is known about metabolic activity of BAT under conditions of fasting and normal temperature. The aim of this study was to compare the possible differences in energetic metabolism between BAT and white adipose tissue (WAT) obtained from rabbits under the conditions of physiological temperature and 24 h after fasting conditions. The study was carried out on New Zealand rabbits (n=10) maintained for a period of 8 weeks at 23±2 °C. Food was removed 24 h before BAT and WAT were obtained. Protein expression levels of the glycolytic-related protein, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate dehydrogenase were higher in WAT than that in BAT. The expression level of carnitine palmitoyltransferase 1 (CPT1) and CPT2, two fatty acid mitochondrial transporters, and the fatty acid β-oxidation-related enzyme, acyl CoA dehydrogenase, was higher in BAT than in WAT. Cytosolic malate dehydrogenase expression and malate dehydrogenase activity were higher in WAT than in BAT. However, lactate dehydrogenase expression and lactate content were significantly higher in BAT than in WAT. In summary, this study for the first time, to our knowledge, has described how under fasting and normal temperature conditions rabbit BAT seems to use anaerobic metabolism to provide energetic fuel, as opposed to WAT, where the malate-aspartate shuttle and, therefore, the gluconeogenic pathway seem to be potentiated.

Effects of factor Xa on the expression of proteins in femoral arteries from type 2 diabetic patients

AIM Further to its pivotal role in haemostasis, factor Xa (FXa) promotes effects on the vascular wall. The purpose of the study was to evaluate if FXa modifies the expression level of energy metabolism and oxidative stress-related proteins in femoral arteries obtained from type 2 diabetic patients with end-stage vasculopathy. METHODS Femoral arteries were obtained from 12 type 2 diabetic patients who underwent leg amputation. Segments from the femoral arteries were incubated in vitro alone and in the presence of 25 nmol l(-1) FXa and 25 nmol l(-1) FXa + 50 nmol l(-1) rivaroxaban. RESULTS In the femoral arteries, FXa increased triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase isotype 1 expression but decreased pyruvate dehydrogenase expression. These facts were accompanied by an increased content of acetyl-CoA. Aconitase activity was reduced in FXa-incubated femoral arteries as compared with control. Moreover, FXa increased the protein expression level of oxidative stress-related proteins which was accompanied by an increased malonyldialdehyde arterial content. The FXa inhibitor, rivaroxaban, failed to prevent the reduced expression of pyruvate dehydrogenase induced by FXa but reduced acetyl-CoA content and reverted the decreased aconitase activity observed with FXa alone. Rivaroxaban + FXa but not FXa alone increased the expression level of carnitine palmitoyltransferase I and II, two mitochondrial long chain fatty acid transporters. Rivaroxaban also prevented the increased expression of oxidative stress-related proteins induced by FXa alone. CONCLUSIONS In femoral isolated arteries from type 2 diabetic patients with end-stage vasculopathy, FXa promoted disruption of the aerobic mitochondrial metabolism. Rivaroxaban prevented such effects and even seemed to favour long chain fatty acid transport into mitochondria.

Differential inhibitory action of apixaban on platelet and fibrin components of forming thrombi: Studies with circulating blood and in a platelet-based model of thrombin generation

Introduction Mechanisms of action of direct oral anticoagulants (DOAC) suggest a potential therapeutic use in the prevention of thrombotic complications in arterial territories. However, effects of DOACs on platelet activation and aggregation have not been explored in detail. We have investigated the effects of apixaban on platelet and fibrin components of thrombus formation under static and flow conditions. Methods We assessed the effects of apixaban (10, 40 and 160 ng/mL) on: 1) platelet deposition and fibrin formation onto a thrombogenic surface, with blood circulating at arterial shear-rates; 2) viscoelastic properties of forming clots, and 3) thrombin generation in a cell-model of coagulation primed by platelets. Results In studies with flowing blood, only the highest concentration of apixaban, equivalent to the therapeutic Cmax, was capable to significantly reduce thrombus formation, fibrin association and platelet-aggregate formation. Apixaban significantly prolonged thromboelastometry parameters, but did not affect clot firmness. Interestingly, results in a platelet-based model of thrombin generation under more static conditions, revealed a dose dependent persistent inhibitory action by apixaban, with concentrations 4 to 16 times below the therapeutic Cmax significantly prolonging kinetic parameters and reducing the total amount of thrombin generated. Conclusions Our studies demonstrate the critical impact of rheological conditions on the antithrombotic effects of apixaban. Studies under flow conditions combined with modified thrombin generation assays could help discriminating concentrations of apixaban that prevent excessive platelet accumulation, from those that deeply impair fibrin formation and may unnecessarily compromise hemostasis.

New strategy of tacrolimus administration in animal model based on tacrolimus-loaded microspheres

New strategies for tacrolimus administration that conserve its immunosuppressive effect but avoiding fluctuations in tacrolimus circulating levels are needed. The aim was to analyze if subcutaneous biodegradable tacrolimus-loaded microspheres injection promoted a significant immunosuppressive response in rats. Rats received two subcutaneous tacrolimus-loaded microspheres injections at different days, the first injection was done at day 0 and the second injection was done 12 days after. Plasma circulating levels of tacrolimus, interleukin-2 (IL-2) and calcineurin phosphatase (PP2B) activity in mononuclear cells were measured. Tacrolimus plasma levels were significantly increased from the day after tacrolimus-loaded microspheres injection and remained increased during 10days. Compared to control, plasma IL-2 levels and PP2B activity in mononuclear cells were significantly decreased during ten days. At day 12, a new subcutaneous injection of tacrolimus-loaded microspheres was performed and two days after injection, tacrolimus plasma levels were again increased and both IL-2 plasma levels and PP2B activity decreased. A single subcutaneous tacrolimus-loaded microspheres injection was enough to reduce tacrolimus-related immunosuppressive parameters. These results open the possibility of new therapeutic strategies to administrate calcineurin inhibitors reducing the variability of their circulating levels related to gastrointestinal drug absorption/metabolism modifications.

Really does temperature reduction and norepinephrine have similar effects on the energy metabolism in rat brown adipose tissue

Abstract Context: Heat generation by brown adipose tissue (BAT) in response to temperature reduction seems to be entirely related to sympathetic nervous stimulation. Objective: To analyse if temperature reduction and norepinephrine may differently affect the expression of proteins related to energy metabolism in BAT. Materials and methods: Isolated rats BAT was incubated with/without norepinephrine (10–6 mol/L, 24 h at 32 °C and 37 °C). Results: In BAT, 32 °C increased the protein expression levels of carnitine palmitoyltransferase-I and -II, mitochondrial uncoupling protein-1 (UCP-1) and the expression and activity of lactate dehydrogenase. Mitochondrial F1-ATP synthase α-chain expression was decreased at 32 °C compared to 37 °C. Norepinephrine and at 32 °C exposure, UCP-1 expression was increased but cytochrome-c oxidase and F1-ATP synthase α-chain expression was reduced with respect to 37 °C. Discussion: Sympathetic stimulation seems not to be the only factor associated with heat generation. Conclusions: Temperature reduction by itself exerts some different effects on the expression of proteins related to the energy metabolism than norepinephrine.