Caveolin and β1-integrin coordinate angiotensinogen expression in cardiac myocytes

Background

The cardiac renin–angiotensin system (RAS) has been implicated in mediating myocyte hypertrophy and remodeling, although the biochemical mechanisms responsible for regulating the local RAS are poorly understood. Caveolin-1 (Cav-1)/Cav-3 double-knockout mice display cardiac hypertrophy, and in vitro disruption of lipid rafts/caveolae using methyl-β-cyclodextrin (MβCD) abolishes cardiac protection.

Methods

In this study, neonatal rat ventricular myocytes (NRVM) were used to determine whether lipid rafts/caveolae may be involved in the regulation of angiotensinogen (Ao) gene expression, a substrate of the RAS system.

Results

Treatment with MβCD caused a time-dependent upregulation of Ao gene expression, which was associated with differential regulation of mitogen-activated protein (MAP) kinases ERK1/2, p38 and JNK phosphorylation. JNK was highly phosphorylated shortly after MβCD treatment (2–30 min), whereas marked activation of ERK1/2 and p38 occurred much later (2–4 h). β_1D-Integrin was required for MβCD-induced activation of the MAP kinases. Pharmacologic inhibition of ERK1/2 and JNK enhanced MβCD-induced Ao gene expression, whereas p38 blockade inhibited this response. Adenovirus-mediated expression of wild-type p38α enhanced MβCD-induced Ao gene expression; conversely expression of dominant negative p38α blocked the stimulatory effects of MβCD. Expression of Cav-3 siRNA stimulated Ao gene expression, whereas overexpression of Cav-3 was inhibitory. Cav-1 and Cav-3 expression levels were found to be positively regulated by p38, but unaffected by ERK1/2 and JNK.

Conclusion

Collectively, these studies indicate that lipid rafts/caveolae couple to Ao gene expression through a mechanism that involves β₁-integrin and the differential actions of MAP kinase family members.     

Prolonged mean reaction time in posterior cerebral artery during visual stimulation in patients with severe carotid stenosis

While the mean increase in flow velocities in posterior cerebral artery (PCA) as a response to visual stimuli is well documented, the data on the reaction time as a measurement of the vasomotor response of the posterior part of the circle of Willis are still sparse. The aim was to assess the visual evoked response in PCA during white light stimulation by means of functional transcranial doppler in patients with severe internal carotid artery (ICA) stenosis, to introduce a real‐time haemodynamic changes as a measurement of the effect of severe carotid disease on the posterior circulation. The measurements were taken in 49 right‐handed patients with severe ICA stenosis or occlusion and 30 healthy volunteers, simultaneously in left and right PCA using 2‐MHz probes, successively in the dark and during the white light stimulation, during three consecutive repetitive periods of 1 min each. Mean values of mean blood flow velocities (MBFV) and mean reaction time (MRT) with and without visual stimuli were analysed. Linear regression analysis showed no statistically significant correlation between the age, MBFV and a degree of left and right carotid stenosis, and MRT in left and right PCA either in the group of healthy subjects or in the group of patients with severe carotid stenosis, in both test conditions. MRT could be an indicator of compromised cerebral circulation in the presence of haemodynamic significant carotid stenosis as well as an additional and independent haemodynamic parameter of the cerebral visual evoked response.     

Limitations of biomarkers serum levels during pulmonary vein isolation

Introduction and objectives

Several biomarkers have been used for evaluation and quantification of myocardial injury after effective ablation. We studied possible different thermal stability and usability of the proteins released by cardiac cells injured by different energy sources.

Methods

Firstly, we tested in vitro thermal stability of creatinine kinase (CK), myocardial bound creatinine kinase (CKMB), cardiac troponins I (cTnI) and cardiac troponins T (cTnT) in collected blood samples from 15 patients (pts) with confirmed ST-segment elevated myocardial infarction (STEMI). Secondly, the biomarkers were collected and analyzed in 82 pts treated with radiofrequency ablation (RFA) and in 79 pts treated with cryo-balloon ablation (CBA).

Results

In vitro experiment showed that all biomarkers were stable in low temperature of -30^oC. Troponins were stable in the high temperatures analyzed. A substantial drop in CK and CKMB levels were measured at 50 °C and 40° C, respectively. In vivo study showed that the increase in CKMB levels was highly significant in CBA pts only. Pathological CKMB values were observed in 24% of RFA pts and 98% of CBA pts. Pathological cTnI values were observed in all pts and the rise in cTnI levels was highly significant in both groups after ablation.

Conclusions

Both in vitro and in vivo results show that CKMB cannot be used for quantitative determination of myocardial injury produced by radiofrequency energy. Only cardiac troponins reflect myocardial injury, regardless of energy source, and may be considered in future studies for comparison of biomarkers effects of cryo versus radiofrequency ablation.Full English text available from: www.revespcardiol.org     

A pro-convulsive carbamazepine metabolite: quinolinic acid in drug resistant epileptic human brain

Drugs and their metabolites often produce undesirable effects. These may be due to a number of mechanisms, including biotransformation by P450 enzymes which are not exclusively expressed by hepatocytes but also by endothelial cells in brain from epileptics. The possibility thus exists that the potency of systemically administered central nervous system therapeutics can be modulated by a metabolic blood-brain barrier (BBB). Surgical brain specimens and blood samples (ex vivo) were obtained from drug-resistant epileptic subjects receiving the antiepileptic drug carbamazepine prior to temporal lobectomies. An in vitro blood-brain barrier model was then established using primary cell culture derived from the same brain specimens. The pattern of carbamazepine (CBZ) metabolism was evaluated in vitro and ex vivo using high performance liquid chromatography-mass spectroscopy. Accelerated mass spectroscopy was used to identify (14)C metabolites deriving from the parent (14)C-carbamazepine. Under our experimental conditions carbamazepine levels could not be detected in drug resistant epileptic brain ex situ; low levels of carbamazepine were detected in the brain side of the in vitro BBB established with endothelial cells derived from the same patients. Four carbamazepine-derived fractions were detected in brain samples in vitro and ex vivo. HPLC-accelerated mass spectroscopy confirmed that these signals derived from (14)C-carbamazepine administered as parental drug. Carbamazepine 10, 11 epoxide (CBZ-EPO) and 10, 11-dihydro-10, 11-dihydrooxy-carbamazepine (DiOH-CBZ) were also detected in the fractions analyzed. (14)C-enriched fractions were subsequently analyzed by mass spectrometry to reveal micromolar concentrations of quinolinic acid (QA). Remarkably, the disappearance of carbamazepine-epoxide (at a rate of 5% per hour) was comparable to the rate of quinolinic acid production (3% per hour). This suggested that quinolinic acid may be a result of carbamazepine metabolism. Quinolinic acid was not detected in the brain of patients who received antiepileptic drugs other than carbamazepine prior to surgery or in brain endothelial cultures obtained from a control patient. Our data suggest that a drug resistant BBB not only impedes drug access to the brain but may also allow the formation of neurotoxic metabolites.     

Lyt phenotypes of primary cytotoxic T cells generated across the A and E region of the H-2 complex

Six different cell-mediated lympholysis (CML) combinations were established, four of which generated effector cells against the I-E, and two against the I-A molecule. The cell surface phenotype of effector cells was then determined by depletion of cytotoxic T lymphocyte (CTL) activity with antisera and rabbit complement (C) treatment. Both types of effector cells were completely eliminated by treatment with anti-Thy-1.2 antiserum plus C. Anti-Lyt-1.2 and C depleted anti-A and anti-E killer activity but did not eliminate CTL generated across a whole H-2 difference. One out of three different batches of anti-Lyt-2.2 antiserum did not deplete anti-A killer activity, while it efficiently eliminated CTL generated across the E region or whole H-2 difference. However, two batches of anti-Lyt-2.2 antiserum depleted also anti-A CTL activity. A quantitative difference between anti-A and anti-E CTL in terms of Lyt-2 expression was demonstrated by significant differences in recovery of killer activity, after treatment of these two types of CTL with a wide concentration range of the same anti-Lyt-2.2 antiserum and C. Thus it is concluded that anti-A killer cells have the cell surface phenotype of Thy-1⁺, Lyt-1, Lyt-2↑, whereas anti-E CTL are Thy-1⁺, Lyt-1↑, Lyt-2←. The data are discussed in the context of a possible association of Lyt phenotypes of T cells with the type of MHC antigens they recognize.     

The efficacy of photon-initiated photoacoustic streaming in the removal of calcium silicate-based filling remnants from the root canal after rotary retreatment

The aim of the study was to evaluate the efficacy of photon-initiated photoacoustic streaming (PIPS) in the removal of filling remnants from root canals after rotary phase of retreatment and to examine the difference in the amount of residual material considering the type of sealer. Thirty-six extracted single-rooted human teeth were instrumented and randomly divided into three groups according to the filling material used: group 1: EndoSequence BC Sealer (Brassler, USA), group 2: MTA Fillapex (Angelus Solucoes Odontologicas, Londrina, Brasil), and group 3: AH Plus sealer (Dentsply DeTrey, Konstanz, Germany). Cold lateral condensation technique was used. After 2 weeks, the root canals were retreated with a rotary phase retreatment system (ProTaper Universal Retreatment, Maillefer, Ballaigues, Switzerland), followed by Er:YAG laser-activated irrigation (photon-initiated photoacoustic streaming, PIPS). The specimens were scanned in a micro-computed tomographic (micro-CT) device after root canal filling, after the rotary retreatment, and after the PIPS. There was significant reduction in the amount of filling material after the rotary phase of retreatment in all groups (p < 0.05), the highest in the MTA Fillapex group (p < 0.001) and no difference between the EndoSequence BC and the AH Plus (p = 0.608). There was significant reduction of the filling remnants after the PIPS in all groups (p < 0.05). The MTA Fillapex was the most easily removed during rotary phase of the retreatment, and there were no differences in the amount of the remaining filling material between EndoSequence BC and the AH Plus groups after rotary phase of the retreatment. The PIPS improved the removal of filling remnants in all groups.