Molecular signatures of atherosclerotic plaques: An up-dated panel of protein related markers.

Atherosclerosis remains the leading cause of ischemic syndromes such as myocardial infarction or brain stroke, mainly promoted by plaque rupture and subsequent arterial blockade. Identification of vulnerable or high-risk plaques constitutes a major challenge, being necessary to identify patients at risk of occlusive events in order to provide them with appropriate therapies. Clinical imaging tools have allowed the identification of certain structural indicators of prone-rupture plaques, including a necrotic lipidic core, intimal and adventitial inflammation, extracellular matrix dysregulation, and smooth muscle cell depletion and micro-calcification. Additionally, alternative approaches focused on identifying molecular biomarkers of atherosclerosis have also been applied. Among them, proteomics has provided numerous protein markers currently investigated in clinical practice. In this regard, it is quite uncertain that a single molecule can describe plaque rupture, due to the complexity of the process itself. Therefore, it should be more accurate to consider a set of markers to define plaques at risk. Herein, we propose a selection of 76 proteins, from classical inflammatory to recently related markers, all of them identified in at least two proteomic studies analyzing unstable atherosclerotic plaques. Such panel could be used as a prognostic signature of plaque instability.

Interplay between lysosomal, mitochondrial and death receptor pathways during manganese-induced apoptosis in glial cells.

Manganese (Mn) is an essential trace metal which plays a critical role in brain physiology by acting as a cofactor for several enzymes. However, upon overexposure, Mn preferentially accumulates within the basal ganglia leading to the development of a Parkinsonism known as Manganism. Data from our group have proved that Mn induces oxidative stress-mediated apoptosis in astrocytoma C6 cells. In the present study we described how cathepsins impact on different steps of each apoptotic cascade. Evidence obtained demonstrated that Mn generates lysosomal membrane permeabilization (LMP) and cathepsin release. Both cathepsins B (Ca-074 Me) and D (Pepstatin A) inhibitors as well as Bafilomycin A1 prevented caspases-3, -7, -8 and -9 activation, FasL upregulation, Bid cleavage, Δφm disruption and cytochrome c release. Results from in vivo studies showed that intrastriatal Mn injection increased cathepsin D levels from corpus striatum and substantia nigra pars compacta. Our results point to LMP and lysosomal cathepsins as key mediators in the apoptotic process triggered by Mn. These findings highlight the relevance of targeting the lysosomal pathway for Manganism therapy.

The autophagic- lysosomal pathway determines the fate of glial cells under manganese- induced oxidative stress conditions.

Manganese (Mn) overexposure is frequently associated with the development of a neurodegenerative disorder known as Manganism. The Mn-mediated generation of reactive oxygen species (ROS) promotes cellular damage, finally leading to apoptotic cell death in rat astrocytoma C6 cells. In this scenario, the autophagic pathway could play an important role in preventing cytotoxicity. In the present study, we found that Mn induced an increase in the amount and total volume of acidic vesicular organelles (AVOs), a process usually related to the activation of the autophagic pathway. Particularly, the generation of enlarged AVOs was a ROS- dependent event. In this report we demonstrated for the first time that Mn induces autophagy in glial cells. This conclusion emerged from the results obtained employing a battery of autophagy markers: a) the increase in LC3-II expression levels, b) the formation of autophagic vesicles labeled with monodansylcadaverine (MDC) or LC3 and, c) the increase in Beclin 1/ Bcl-2 and Beclin 1/ Bcl-X(L) ratio. Autophagy inhibition employing 3-MA and mAtg5(K130R) resulted in decreased cell viability indicating that this event plays a protective role in Mn- induced cell death. In addition, mitophagy was demonstrated by an increase in LC3 and TOM-20 colocalization. On the other hand, we proposed the occurrence of lysosomal membrane permeabilization (LMP) based in the fact that cathepsins B and D activities are essential for cell death. Both cathepsin B inhibitor (Ca-074 Me) or cathepsin D inhibitor (Pepstatin A) completely prevented Mn- induced cytotoxicity. In addition, low dose of Bafilomycin A1 showed a similar effect, a finding that adds evidence about the lysosomal role in Mn cytotoxicity. Finally, in vivo experiments demonstrated that Mn induces injury and alters LC3 expression levels in rat striatal astrocytes. In summary, our results demonstrated that autophagy is activated to counteract the harmful effect caused by Mn. These data is valuable to be considered in future research concerning Manganism therapies.

A redox-based mechanism for the contractile and relaxing effects of NO in the guinea-pig gall bladder.

The purpose of this study was to determine the effects of sodium nitroprusside (SNP), 2,2'-(hydroxynitrosohydrazino)bis-ethanamine (DETA/NO) and 3-morpholinosydnonimine (SIN-1), NO donors which yield different NO reactive species (NO+, NO* and peroxynitrite, respectively), as well as exogenous peroxynitrite, on gall bladder contractility. Under resting tone conditions, SNP induced a dose-dependent contraction with a maximal effect (10.3 +/- 0.7 mN, S.E.M.) at 1 mM. Consistent with these findings, SNP caused a concentration-dependent depolarization of gall bladder smooth muscle. The excitatory effects of SNP were dependent on extracellular calcium entry through L-type Ca2+ channels. Furthermore, the contraction and depolarization were sensitive to tyrosine kinase blockade, and an associated increase in tyrosine phosphorylation was detected in Western blot studies. DETA/NO induced dose-dependent relaxing effects. These relaxations were sensitive to the guanylyl cyclase inhibitor 1H-[1,2,4]oxidiazolo[4,3-a]quinoxaline-1-one (ODQ, 2 microM) but they were not altered by treatment with the potassium channel blockers tetraethylammoniun (TEA, 5 mM) and 4-aminopyridine (4-AP, 5 mM). When tested in a reducing environment (created by 2.5 mM 1,4-dithiothreitol, DTT), SNP caused a relaxation of gall bladder muscle strips. Similarly, the SNP-induced contraction was converted to a relaxation, and associated hyperpolarization, when DTT was added during the steady state of an SNP-induced response. SIN-1 (0.1 mM), which has been shown to release peroxynitrite, induced relaxing effects that were enhanced by superoxide dismutase (SOD, 50 U ml(-1)). The relaxations induced by either SIN-1 alone or SIN-1 in the presence of SOD were strengthened by catalase (1000 U ml(-1)) and abolished by ODQ pretreatment. However, exogenous peroxynitrite induced a concentration-dependent contraction, which was dependent on activation of leukotriene (LT) metabolism and extracellular calcium. The peroxynitrite-induced contraction was abolished in the presence of the peroxynitrite scavenger melatonin. These results suggest that SIN-1 behaves as an NO* rather than a peroxynitrite source. We conclude that, depending on the redox state, NO has opposing effects on the motility of the gall bladder, being a relaxing agent when in NO * form and a contracting agent when in NO+ or peroxynitrite redox species form. Knowledge of the contrasting effects of the different redox forms of NO can clarify our understanding of the effects of NO donors on gall bladder and other smooth muscle cell types.

Relaxation of canine gallbladder to nerve stimulation involves adrenergic and non-adrenergic non-cholinergic mechanisms.

Electrical field stimulation (EFS) of dog gallbladder strips induced a frequency-dependent contractile response followed by an off-relaxation that was turned into a pure inhibitory response after atropine pretreatment. Guanethidine reduced the atropine-induced relaxing responses, so an adrenergic mechanism can partially account for the nerve-mediated gallbladder relaxation. However, guanethidine pretreatment also revealed a nonadrenergic noncholinergic (NANC) relaxation induced by EFS, which was frequency independent. NANC relaxations were reduced by L-arginine methyl ester (L-NAME, 100 micromol L-1), a nitric oxide synthase inhibitor (D-p-Cl-Phe6, Leul7; 10 micromol L-1), a vasoactive intestinal peptide (VIP) receptor antagonist, and an inhibitor of haem oxygenase, (copper protoporphyrin IX; CuPP-IX; 10 micromol L-1), suggesting that nitric oxide (NO), VIP and carbon monoxide (CO), respectively, are released in response to EFS. Immunoreactivities for haem oxygenase-2 (HO-2) and VIP, and histochemical staining for NADPH diaphorase were observed in nerve cell bodies and fibres, demonstrating the presence of CO, VIP and NO as putative NANC neurotransmitters in dog gallbladder. These data support the hypothesis that NO, VIP and CO contribute to NANC relaxation of the canine gallbladder.

Activation of tyrosine kinase pathway by vanadate in gallbladder smooth muscle.

Vanadate, an inhibitor of tyrosine phosphatase activity, might induce gallbladder contraction through the stimulation of the tyrosine kinase pathway. The aim of this study was to characterize the effects of vanadate in the guinea pig gallbladder smooth muscle. Vanadate exerts contractile effects which are not mediated by neurotransmitter release. The tyrosine kinase inhibitor genistein nearly abolished vanadate contraction, suggesting that an increase in protein tyrosine phosphorylation mediates the actions of vanadate. This suggestion was confirmed by Western blot analysis. Vanadate contractions were reduced in the presence of methoxyverapamil or in Ca(2+)-free medium, suggesting that vanadate may induce Ca(2+) influx. Neither inactivation of the Na(+)/K(+) pump nor reversal of the Na(+)/Ca(2+) exchanger can account for vanadate's actions. Vanadate contractile effects were reduced by indomethacin, as well as mepacrine, the inhibitor of phospholipase A(2), but were not affected by phospholipase C inhibitors. Neither inhibitors of diacylglycerol lipase nor protein kinase C reduced the response induced by vanadate. These data indicate that the effects of vanadate on smooth muscle are mainly mediated by protein tyrosine phosphorylation and reveal a new link between tyrosine phosphorylation and arachidonic acid metabolism in the control of gallbladder smooth muscle contraction.

Secretin potentiates guinea pig pancreatic response to cholecystokinin by a cholinergic mechanism.

The effects of secretin and cholecystokinin on exocrine pancreas secretion in the guinea pig were investigated. The putative potentiating effect of these two hormones was studied in various settings to elucidate the effect of cholinergic stimuli in such interaction. In anesthetized guinea pig, intravenous infusion of cholecystokinin (0.75 pmol.kg-1.min-1) or secretin (0.5 pmol.kg-1.min-1) resulted in a marked and rapid increase of pancreatic juice flow and protein output. When cholecystokinin was combined with secretin, there was a significant increase in pancreatic, compared with cholecystokinin alone. This increase in pancreatic juice secretion and protein output was significantly suppressed by the prior administration of 100 micrograms/kg atropine. Similar results were obtained when trypsinogen release from pancreatic segments was measured in response to cholecystokinin (32 nM-32 pM) and (or) secretin (1 microM-32 nM). When we assayed the hormonal interaction on amylase release from dispersed pancreatic acini, we found that secretin (32 nM) failed to influence the secretory response to cholecystokinin (1 pM-10 nM). Thus we conclude that a combination of cholecystokinin and secretin resulted in a marked potentiation of the secretory responses in the exocrine guinea pig pancreas by a mechanism that involves cholinergic interactions present at the tissue level but not at the dispersed secretory cell level.

Cholinergic dependence of pancreatic response to cholecystokinin in rats and guinea pigs.

1. Lorglumide and atropine were used to examine the role of cholinergic mechanisms in the pancreatic secretory response to cholecystokinin in two animal species. 2. Anaesthetized rats and guinea pigs with jugular vein and pancreatic cannulae were used and the bile juice was recirculated. In the rat, the treatment with lorglumide (3 mumol/kg) as well as atropine (100 micrograms/kg) did not have effects on basal interdigestive secretion, whereas in guinea pigs only atropine decreased the protein output (41%) and the juice flow (47%) of the basal pancreatic secretion. 3. Infusion of cholecystokinin (150 pmol/kg/hr in the rat and 50 pmol/kg/hr in the guinea pig) induced a marked increase in pancreatic juice flow and protein output compared to saline controls. Pretreatment of both rat and guinea pig with lorglumide resulted in a marked attenuation of the cholecystokinin-evoked secretory response. 4. In the rats, atropine decreased the response to infusion of cholecystokinin octapeptide while this antimuscarinic agent had no effect in the response to cholecystokinin in the guinea pigs. 5. This study supports the concept that the influence of cholinergic system in pancreatic response to cholecystokinin shows interspecific differences.

Histaminergic modulation of hormonal control in the exocrine guinea-pig pancreas.

The effects of histamine upon secretin- or cholecystokinin (CCK)-evoked exocrine pancreatic secretion were investigated in the anaesthetised guinea pig. Histamine (0.1 mumol/kg/min) induced a slight increase in pancreatic juice flow and total protein release compared to saline controls. Secretin (0.5 pmol/kg/min) and CCK-8 (0.75 pmol/kg/min) evoked marked time course increases in both the rate of pancreatic juice flow and total protein output in the anaesthetised guinea pig. Administration of either secretin or CCK-8 simultaneously with histamine elevated the exocrine pancreatic secretion compared to the smaller response obtained when administered separately. These results indicate that histamine may play an important physiological role in modulating the hormonal control of exocrine guinea pig pancreas.