Rafaela da Silva

Preconditioning by Sevoflurane Decreases Biochemical Markers for Myocardial and Renal Dysfunction in Coronary Artery Bypass Graft Surgery: A Double-blinded, Placebo-controlled, Multicenter Study

Background Preconditioning by volatile anesthetics is a promising therapeutic strategy to render myocardial tissue resistant to perioperative ischemia. It was hypothesized that sevoflurane preconditioning would decrease postoperative release of brain natriuretic peptide, a biochemical marker for myocardial dysfunction. In addition, several variables associated with the protective effects of preconditioning were evaluated. Methods Seventy-two patients scheduled for coronary artery bypass graft surgery under cardioplegic arrest were randomly assigned to preconditioning during the first 10 min of complete cardiopulmonary bypass with either placebo (oxygen–air mixture only) or sevoflurane 4 vol% (2 minimum alveolar concentration). No other volatile anesthetics were administered at any time during the study. Treatment was strictly blinded to anesthesiologists, perfusionists, and surgeons. Biochemical markers of myocardial dysfunction and injury (brain natriuretic peptide, creatine kinase–MB activity, and cardiac troponin T), and renal dysfunction (cystatin C) were determined. Results of Holter electrocardiography were recorded perioperatively. Translocation of protein kinase C was assessed by immunohistochemical analysis of atrial samples. Results Sevoflurane preconditioning significantly decreased postoperative release of brain natriuretic peptide, a sensitive biochemical marker of myocardial contractile dysfunction. Pronounced protein kinase C &dgr; and &egr; translocation was observed in sevoflurane-preconditioned myocardium. In addition, postoperative plasma cystatin C concentrations increased significantly less in sevoflurane-preconditioned patients. No differences between groups were found for perioperative ST-segment changes, arrhythmias, or creatine kinase–MB and cardiac troponin T release. Conclusions Sevoflurane preconditioning preserves myocardial and renal function as assessed by biochemical markers in patients undergoing coronary artery bypass graft surgery under cardioplegic arrest. This study demonstrated for the first time translocation of protein kinase C isoforms &dgr; and &egr; in human myocardium in response to sevoflurane.

Translocation of Protein Kinase C Isoforms to Subcellular Targets in Ischemic and Anesthetic Preconditioning

Background Translocation of protein kinase C (PKC) to subcellular targets is a pivotal signaling step in ischemic preconditioning (IPC). However, to date, it is unknown whether PKC isoforms translocate in anesthetic preconditioning (APC). Methods The PKC blockers chelerythrine and rottlerin and the adenosine triphosphate–dependent potassium (KATP) channel blockers HMR-1098 and 5-hydroxydecanoate were used to assess the role of PKC and KATP channels in isolated perfused rat hearts subjected to IPC or APC (1.5 minimum alveolar concentration isoflurane) followed by 40 min of ischemia and 30 min of reperfusion. Immunohistochemical techniques were used to visualize PKC translocation after preconditioning. In addition, the phosphorylation status of PKC isoforms was assessed. Results Chelerythrine, rottlerin, and 5-hydroxydecanoate blocked IPC and APC with respect to functional recovery, albeit IPC at higher concentrations. HMR-1098 did not affect IPC or APC. PKC&dgr; and PKC&egr; translocated to nuclei in both IPC and APC, which was inhibited by chelerythrine and rottlerin. PKC&dgr; translocated to mitochondria but not to the sarcolemma, and PKC&egr; translocated to the sarcolemma and intercalated disks but not to mitochondria. Interestingly, PKC&egr; was accumulated at the intercalated disks in control and preconditioned hearts. Phosphorylation of PKC&dgr; on serine643 was increased in IPC and APC and blocked by chelerythrine and rottlerin, whereas phosphorylation of PKC&dgr; on threonine505 was increased only in IPC and not blocked by chelerythrine or rottlerin. PKC&egr; on serine729 did not change its phosphorylation status. Conclusions This study indicates that translocation of PKC&dgr; plays a pivotal role in IPC and APC and suggests that phosphorylation of PKC&dgr; on serine643 may be of particular relevance in transferring the APC stimulus to mitochondrial KATP channels.

Trigger-dependent gene expression profiles in cardiac preconditioning: evidence for distinct genetic programs in ischemic and anesthetic preconditioning.

BackgroundDNA chips facilitate genomic-wide exploration of gene expression. The authors hypothesized that ischemic (IPC) and anesthetic preconditioning (APC) would differentially modulate gene expression in hearts. MethodsAffymetrix rat U34A gene chips were used to explore the transcriptional response to IPC and APC, sustained ischemia (110 min) without reperfusion, and time-matched perfusion in isolated rat hearts. IPC was induced by three cycles of 5 min of ischemia, and APC was induced by 1.5 minimum alveolar concentration isoflurane (110 min). For each heart, a separate chip was used for hybridization. Data were analyzed for significant ≥ 2.0-fold changes in gene expression. Microarray results were confirmed by quantitative real-time reverse-transcription polymerase chain reaction. ResultsOf the 8,799 genes represented on U34A, 217 transcripts in the APC group, 234 in the IPC group, and 29 in the ischemia group displayed significant ≥ 2.0-fold up-regulation in messenger RNA levels, and 185 transcripts in the APC group, 55 in the IPC group, and 49 in the ischemia group displayed significant ≥ 2.0-fold down-regulation. Many of these transcripts were unknown genes. A high number of commonly regulated genes were found in IPC and APC (39 up-regulated, 17 down-regulated). Genes commonly regulated included those associated with cell defense (heat shock protein 10, aldose reductase, Bcl-xS). Conversely, a pool of protective and antiprotective genes was differentially regulated in APC versus IPC (heat shock protein 27/70, programmed cell death 8), suggesting trigger-dependent transcriptome variability. ConclusionsThe novel microarray technology provides evidence for distinct cardioprotective phenotypes in IPC and APC. The observed transcriptional changes raise the possibility of a second window of protection by volatile anesthetics. The authors’ molecular portraits are the first global genomic comparison between IPC and APC.

Differential Activation of Mitogen-activated Protein Kinases in Ischemic and Anesthetic Preconditioning

BackgroundAccumulating evidence pinpoints to the pivotal role of mitogen-activated protein kinases (MAPKs) in the signal transduction underlying cardiac preconditioning. MethodsPD98059, an inhibitor of extracellular signal–regulated protein kinase (MEK-ERK1/2), and SB203580, an inhibitor of p38 MAPK, were used to evaluate the role of MAPKs with respect to postischemic functional recovery in isolated perfused rat hearts subjected to ischemic preconditioning (IPC) and anesthetic preconditioning (APC). Western blot analyses were used to determine the degree of ERK1/2 and p38 MAPK activation after the application of the preconditioning stimulus and after ischemia–reperfusion. Immunohistochemical staining served to visualize subcellular localization of activated MAPKs. ResultsPD98059 and SB203580 abolished postischemic functional recovery in IPC but not in APC. IPC but not APC markedly activated ERK1/2 and p38 MAPK, which were abrogated by coadministration of the specific blockers. Conversely, IPC and APC enhanced ERK1/2 activity after ischemia–reperfusion as compared to nonpreconditioned hearts, and IPC in addition enhanced p38 MAPK activity. Coadministration of PD98059 and SB203580 during IPC but not during APC inhibited postischemically enhanced MAPK activities. Moreover, chelerythrine and 5-hydroxydecanoate, effective blockers of IPC and APC, annihilated IPC- and APC-induced enhanced postischemic responses of MAPKs. Finally, administration of PD98059 during ischemia–reperfusion diminished the protective effects of IPC and APC. Immunohistochemistry revealed increased ERK1/2 activity primarily in intercalated discs and nuclei and increased p38 MAPK activity in the sarcolemma and nuclei of IPC-treated hearts. ConclusionsAlthough MAPKs may orchestrate cardioprotection as triggers and mediators in IPC, they are devoid of triggering, but they may have mediator effects in APC.

The activation of the cannabinoid receptor type 2 reduces neutrophilic protease-mediated vulnerability in atherosclerotic plaques

AIMS The activation of cannabinoid receptor type 2 (CB(2))-mediated pathways might represent a promising anti-atherosclerotic treatment. Here, we investigated the expression of the endocannabinoid system in human carotid plaques and the impact of CB(2) pharmacological activation on markers of plaque vulnerability in vivo and in vitro. METHODS AND RESULTS The study was conducted using all available residual human carotid tissues (upstream and downstream the blood flow) from our cohort of patients symptomatic (n = 13) or asymptomatic (n = 27) for ischaemic stroke. Intraplaque levels of 2-arachidonoylglycerol, anandamide N-arachidonoylethanolamine, N-palmitoylethanolamine, N-oleoylethanolamine, and their degrading enzymes (fatty acid amide hydrolase and monoacylglycerol lipase) were not different in human plaque portions. In the majority of human samples, CB(1) (both mRNA and protein levels) was undetectable. In downstream symptomatic plaques, CB(2) protein expression was reduced when compared with asymptomatic patients. In these portions, CB(2) levels were inversely correlated (r = -0.4008, P = 0.0170) with matrix metalloprotease (MMP)-9 content and positively (r = 0.3997, P = 0.0174) with collagen. In mouse plaques, CB(2) co-localized with neutrophils and MMP-9. Treatment with the selective CB(2) agonist JWH-133 was associated with the reduction in MMP-9 content in aortic root and carotid plaques. In vitro, pre-incubation with JWH-133 reduced tumour necrosis factor (TNF)-α-mediated release of MMP-9. This effect was associated with the reduction in TNF-α-induced ERK1/2 phosphorylation in human neutrophils. CONCLUSION Cannabinoid receptor type 2 receptor is down-regulated in unstable human carotid plaques. Since CB(2) activation prevents neutrophil release of MMP-9 in vivo and in vitro, this treatment strategy might selectively reduce carotid vulnerability in humans.

Role of Renin-Angiotensin System in Inflammation, Immunity and Aging

Recent data support the idea that the effects of RAS are not restricted to the cardiovascular and renal systems. Importantly, RAS modulates free radical production and the cellular synthesis of several molecules such as cytokines, chemokines and transcription factors. These functions reflect directly the RAS ability to modulate the cell growth, senescence and migration. Activation of the classic RAS, ACE/Ang II/AT1R, has been strictly related to down regulation of pro-survival genes (Nampt and Sirt3), increase in ROS production and pro-inflammatory cytokines and chemokines release, leading to cell senescence, inflammation and development of autoimmune dysfunctions. However, the new view of RAS, points to the ACE2/Ang-(1-7)/Mas receptor axis as a counter-regulator of the effects of the classic Ang II-mediated effects. This new pathway is not totally elucidated. However, some studies suggest an important role of this novel axis in the control of cytokines release as well as cell migration and synthesis, preventing extra-cellular matrix deposition and cell apoptosis. Classic RAS blockers have been proposed as anti-inflammatory and immunomodulatory agents and some studies suggest a new potential application of RAS blockers in autoimmune diseases. The aim of the present review is to update the novel roles of classical and new RAS components and their possible implication during the physiological aging, in the immune system and inflammation.

Recombinant tissue plasminogen activator induces blood–brain barrier breakdown by a matrix metalloproteinase-9-independent pathway after transient focal cerebral ischemia in mouse

The role of the inducible matrix metalloproteinase (MMP)‐9 in blood–brain barrier (BBB) disruption after ischemic stroke is well accepted. Recombinant tissue plasminogen activator (r‐tPA) is the only approved thrombolytic treatment of ischemic stroke but r‐tPA is potentially neurotoxic. Vasogenic edema after r‐tPA treatment has been linked with an increase in cerebral MMP‐9. However, because cerebral ischemia clearly increases the levels of endogenous tPA, the consequence of additional r‐tPA may be questionable. In this study, wild type and MMP‐9 knockout mice were subjected to 90 min transient middle cerebral artery occlusion and treated with 10 mg/kg r‐tPA. At 24 h after occlusion, BBB permeability, hemispheric enlargement, collagen and laminin degradation as well as cerebral infarction were increased in both wild type and MMP‐9 knockout treated animals as compared with non‐treated animals. Mortality was increased in wild type but reduced in knockout treated mice. Cerebral MMP‐9 concentration was not modified by r‐tPA. However, pre‐treatment with p‐aminobenzoyl‐gly‐pro‐D‐leu‐D‐ala‐hydroxamate, a broad‐spectrum MMP inhibitor, counteracted the effects of r‐tPA on the neurovascular unit and decreased mortality in both wild type and knockout mice. MMP inhibition did not modify cerebral infarction in r‐tPA‐treated animals. Our results suggest that r‐tPA toxicity is mainly independent of MMP‐9 after transient middle cerebral artery occlusion but could involve some other MMPs. Additionally, our results support the hypothesis of a dissociation between r‐tPA‐dependent mechanisms of BBB breakdown and cerebral infarction. Due to the importance of r‐tPA in thrombolytic treatment of ischemic stroke patients, the MMPs that could participate in r‐tPA‐induced BBB disruption should be further characterized.

An oral formulation of angiotensin-(1-7) reverses corpus cavernosum damages induced by hypercholesterolemia.

INTRODUCTION The renin angiotensin system plays a crucial role in erectile function. It has been shown that elevated angiotensin-II levels contribute to the development of erectile dysfunction (ED). Oppositely, angiotensin-(1-7) (Ang-[1-7]) mediates penile erection by activation of receptor Mas. Recently, we have developed a formulation based on Ang-(1-7) inclusion in cyclodextrin (CyD) [Ang-(1-7)-CyD], which allows for the oral administration of Ang-(1-7). AIM In the present study, we evaluated the effects of chronic treatment with Ang-(1-7)-CyD on penile fibrosis, oxidative stress, and endothelial function in hypercholesterolemic mice. METHODS Apolipoprotein(Apo)E-/- mice fed a Western-type diet for 11 weeks received Ang-(1-7)-CyD or vehicle during the final 3 weeks. Collagen content and reactive oxygen species (ROS) production within the corpus cavernosum were evaluated by Sirius red and dihydroethidium staining, respectively. Protein expression of neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS), nicotinamide adenine dinucleotide phosphate (NADPH) subunits (p67-phox and p22-phox), and AT1 and Mas receptors in the penis was assessed by Western blotting. Nitric oxide (NO) production was measured by Griess assay in the mice serum. Cavernosal strips were mounted in an isometric organ bath to evaluate the endothelial function. MAIN OUTCOME MEASURES The effect of Ang-(1-7)-CyD treatment on penile fibrosis, oxidative stress, and endothelial function in hypercholesterolemia-induced ED. RESULTS Ang-(1-7)-CyD treatment reduced collagen content in the corpus cavernosum of ApoE-/- mice. This effect was associated with an attenuation of ROS production and a diminished expression of NADPH. Furthermore, Ang-(1-7)-CyD treatment augmented the expression of nNOS and eNOS in the penis and elevated vascular NO production. Importantly, these effects were accompanied by an improvement in cavernosal endothelial function. CONCLUSION Long-term treatment with Ang-(1-7)-CyD reduces penile fibrosis associated with attenuation of oxidative stress. Additionally, cavernosal endothelial function in hypercholesterolemic mice was markedly improved. These results suggest that Ang-(1-7)-CyD might have significant therapeutic benefits for the treatment of erectile dysfunction.

Update on the Role of Cannabinoid Receptors after Ischemic Stroke

Cannabinoids are considered as key mediators in the pathophysiology of inflammatory diseases, including atherosclerosis. In particular, they have been shown to reduce the ischemic injury after acute cardiovascular events, such as acute myocardial infarction and ischemic stroke. These protective and anti-inflammatory properties on peripheral tissues and circulating inflammatory have been demonstrated to involve their binding with both selective cannabinoid type 1 (CB1) and type 2 (CB2) transmembrane receptors. On the other hands, the recent discoveries of novel different classes of cannabinoids and receptors have increased the complexity of this system in atherosclerosis. Although only preliminary data have been reported on the activities of novel cannabinoid receptors, several studies have already investigated the role of CB1 and CB2 receptors in ischemic stroke. While CB1 receptor activation has been shown to directly reduce atherosclerotic plaque inflammation, controversial data have been shown on neurotransmission and neuroprotection after stroke. Given its potent anti-inflammatory activities on circulating leukocytes, the CB2 activation has been proven to produce protective effects against acute poststroke inflammation. In this paper, we will update evidence on different cannabinoid-triggered avenues to reduce inflammation and neuronal injury in acute ischemic stroke.

Increase of arginase activity in old apolipoprotein-E deficient mice under Western diet associated with changes in neurovascular unit.

Aging and atherosclerosis are well-recognized risk factors for cardiac and neurovascular diseases. The Apolipoprotein E deficient (ApoE−/−) mouse on a high-fat diet is a classical model of atherosclerosis, characterized by the presence of atherosclerotic plaques in extracranial vessels but not in cerebral arteries. Increase in arginase activity was shown to participate in vascular dysfunction in the peripheral arteries of atherosclerotic mice by changing the level of nitric oxide (NO). NO plays a key role in the physiological functions of the neurovascular unit (NVU). However, the regulation of arginase expression and activity in the brain was never investigated in association with changes in the NVU, ApoE deficiency and high fat diet.Fourteen-month-old ApoE−/− mice on high-fat diet exhibited deposition of lipids in the NVU, impairment of blood–brain barrier properties, astrogliosis and an increase of aquaporin 4 staining. In association with these changes, brain arginase activity was significantly increased in the old ApoE−/− mice as compared to old wild type mice, with an increase in the level of arginase type I in the blood vessels.In conclusion, aging in this classical mouse model of atherosclerosis induces an increase in the level and activity of arginase I that may impair NO synthesis and contribute to changes in the NVU leading to blood–brain barrier leakage and inflammation.