Time course of necrosis/apoptosis and neovascularization during experimental gastric conditioning.

Apoptosis, necrosis and neovascularization are three processes that occur during ischemic preconditioning in a range of organs. In the stomach, the effect of this preconditioning (the delay phenomenon) has helped to improve gastric vascularization prior to esophagogastric anastomosis after esophagectomy. Here we present a sequential study of the histological recovery of the gastric fundus and the phenomena of apoptosis, necrosis and neovascularization in an experimental model of partial gastric ischemia. Partial gastric devascularization was performed by ligature of the left gastric vessels in Sprague-Dawley rats. Rats were assigned to groups in accordance with their evaluation period: control, 1, 3, 6, 10, 15 and 21 days. Histological analysis, caspase-3 activity, DNA fragmentation and vascular endothelial cell proliferation (Ki-67) were measured in tissue samples after sacrifice. After 24 h of partial gastric ischemia, rates of apoptosis and necrosis were higher in the experimental groups than in controls. Tissue injury was higher 3 and 6 days post-ischemia. From day 10 after partial gastric ischemia, apoptosis and necrosis started to decrease, and on days 15 and 21 showed no differences in relation to controls. Neovascularization began between days 1 and 3, reaching its peak at 15 days after ischemia and coinciding with complete histological recovery. Both necrosis and apoptosis play a role in tissue injury during the first days after partial gastric ischemia. After 15 days, the evolution of both the histology and the neovascularization suggested that this is the optimal time for performing gastric transposition.

Effects of interferon-gamma on nitric oxide synthase activity and endothelin-1 production by vascular endothelial cells.

Given the pivotal role suggested for IFN-gamma in immune diseases of the vascular wall, we investigated the effects of IFN-gamma on nitric oxide (NO) and endothelin-1 (ET-1) expression in bovine aortic endothelial cells (BAEC). We have previously reported that TNF-alpha enhanced NO synthase activity in BAEC as assessed by quantifying release of bioactive NO with reporter monolayers and measuring conversion of L-[14C]arginine to L-[14C] citrulline. In murine macrophages IFN-gamma synergizes with TNF-alpha or lipopolysaccharide to induce robust increases in calcium-independent NO synthase activity. In this study we have found that IFN-gamma alone failed to have a significant effect on NO synthase activity in BAEC. In contrast to murine macrophages, IFN-gamma inhibited TNF-alpha-stimulated induction of endothelial NO synthase activity in a concentration-dependent manner. This observation suggests that there is major difference in the response of BAEC and murine macrophages to IFN-gamma. A second major aim of this study was to determine the effect of IFN-gamma on preproET-1 mRNA expression and ET-1 secretion rates in BAEC. IFN-gamma alone had little or no effect on ET-1 mRNA levels and basal ET release when measured for 8 h. However, cotreatment with IFN-gamma potentiated the stimulatory effect of TNF-alpha on BAEC ET-1 mRNA transcript levels and ET release. In contrast, pretreatment of cells with IFN-gamma for 16-24 h blunted the stimulatory effect of TNF-alpha. These findings suggest that endothelial cell expression of vasoactive mediators is modified by the temporal interplay of at least two immune mediators, IFN-gamma and TNF-alpha.

Effects of the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, atorvastatin and simvastatin, on the expression of endothelin-1 and endothelial nitric oxide synthase in vascular endothelial cells.

Endothelial dysfunction associated with atherosclerosis has been attributed to alterations in the L-arginine-nitric oxide (NO)-cGMP pathway or to an excess of endothelin-1 (ET-1). The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have been shown to ameliorate endothelial function. However, the physiological basis of this observation is largely unknown. We investigated the effects of Atorvastatin and Simvastatin on the pre-proET-1 mRNA expression and ET-1 synthesis and on the endothelial NO synthase (eNOS) transcript and protein levels in bovine aortic endothelial cells. These agents inhibited pre-proET-1 mRNA expression in a concentration- and time-dependent fashion (60-70% maximum inhibition) and reduced immunoreactive ET-1 levels (25-50%). This inhibitory effect was maintained in the presence of oxidized LDL (1-50 microg/ml). No significant modification of pre-proET-1 mRNA half-life was observed. In addition, mevalonate, but not cholesterol, reversed the statin-mediated decrease of pre-proET-1 mRNA levels. eNOS mRNA expression was reduced by oxidized LDL in a dose-dependent fashion (up to 57% inhibition), whereas native LDL had no effect. Statins were able to prevent the inhibitory action exerted by oxidized LDL on eNOS mRNA and protein levels. Hence, these drugs might influence vascular tone by modulating the expression of endothelial vasoactive factors.

Paradoxical recovery in a bilingual patient with aphasia after right capsuloputaminal infarction.

We report the case of a bilingual dextral patient, who presented with an uncommon pattern of aphasic deficit following a right capsulo-putaminal infarction. In this patient, the linguistic deficit concerned the use of her mother tongue (Galician, L1) much more than the lesser practised second language (Spanish, L2). Our patient presented spontaneous fluent speech in L2 but not in L1, automatic translation into L2, and impaired repetition in L1, whereas comprehension was spared in both L1 and L2. Reading and writing were less valuable due to educational interference (reduced schooling). Spontaneous speech 16 months after the stroke showed the stability of the impairment. This is the first reporting of a crossed subcortical aphasia in a bilingual patient.

Testing the use of juvenile Salmo trutta L. as biomonitors of heavy metal pollution in freshwater.

Individual specimens of Salmo trutta were captured, from four sampling sites in Galician rivers (NW Spain) affected by different types of contamination: diffuse urban waste, run-off from an unrestored dump at a copper mine and waste from a fish farm. The ages of the captured trouts were established and only those belonging to the 1+ age class were selected for study. The liver and kidney were removed from each fish and analysed to determine the tissue concentrations of Cu, Fe and Zn. The results obtained showed that: (i) the use of 1+ individuals allowed differentiation of contamination scenarios on the basis of the tissue concentrations of metal; (ii) the use of 1+ individuals allowed standardization of the time of exposure, which was sufficiently long for differential uptake to have taken place; (iii) liver tissue provided the best results as, less effort was required than for processing kidney tissue, and significant differences between sampling sites were detected because the intrapopulational variability in metal levels was lower than for kidney, and (iv) the levels of elements detected were not affected by basal tissue concentrations or residual concentrations due to past contamination, which older trouts may have been exposed to. In addition, the use of 1+ trout may provide better results in annual environmental sampling surveys.

Involvement of Rho GTPases in the transcriptional inhibition of preproendothelin-1 gene expression by simvastatin in vascular endothelial cells.

Endothelial dysfunction is characterized by an impaired vasodilatory response to endothelial agonists as well as by alterations in adhesion and coagulation processes. 3-Hydroxy-3-methylglutaryl-CoA reductase inhibitors (statins) have been shown to be useful in the reversal of endothelial dysfunction, an effect that may be independent of the reduction in cholesterol levels. Both the L-arginine-nitric oxide-cGMP and endothelin pathways are involved in the regulation of vascular tone. Here, we show that the basal transcription rate of the preproendothelin-1 gene was decreased by simvastatin (10 micromol/L) in bovine aortic endothelial cells. Transfection studies with the preproendothelin-1 gene promoter showed that mevalonate (100 micromol/L) was able to prevent the inhibitory effect mediated by simvastatin. Protein geranylgeranylation, but not farnesylation, proved to be crucial for a correct expression of the preproendothelin-1 gene. The C3 exotoxin from Clostridium botulinum that selectively inactivates Rho GTPases, the processing of which involves geranylgeranylation, reproduced the inhibitory effect of simvastatin on the expression of preproendothelin-1. Overexpression of dominant-negative mutants of RhoA and RhoB led to a significant reduction in the preproendothelin-1 promoter activity, whereas the expression of wild-type and constitutively active forms of these proteins resulted in an increase, in support that Rho proteins are required for the basal expression of the preproendothelin-1 gene. Finally, we show that the Rho-dependent activation of the preproendothelin-1 gene transcription was inhibited by simvastatin. Thus, the control of vascular tone and proliferative response mediated by endothelin-1 is regulated at multiple levels, among which the Rho proteins play an essential role.

Oxidative and nitrosative stress in kidney disease: a case for cyclosporine A.

The immunosuppresor cyclosporine A (CsA) has been associated to human endothelial dysfunction and accelerated atherosclerosis. Sympathetic overactivity, relative deficiency of nitric oxide, TGFb-1, endothelin-1, reactive oxygen (ROS) and nitrogen species (RNS) and vasoconstrictor eicosanoids are mediators of vascular dysfunction associated to cyclosporine A. In CsA-treated cells (BAEC) an increase in reactive oxygen and nitrogen intermediates may lead to the intracellular formation of peroxynitrite. This agent could be one important mediator by which CsA produces an antioxidant-sensitive nitration of tyrosine, a marker for endothelial damage by nitrosative stress. Superoxide anion is the limiting factor in the formation of peroxynitrite in CsA-treated endothelial cells. Treatment with CsA may lead to the nitration of specific proteins such as manganese superoxide dismutase (MnSOD). We propose that peroxynitrite and tyrosine nitration may represent mechanisms of damage in pathophysiological situations where superoxide anion generation is increased.

Native and oxidized low density lipoproteins oppositely modulate the effects of insulin-like growth factor I on VSMC.

OBJECTIVE: Changes in the local expression and signaling activity of the insulin-like growth factor-I (IGF-I) axis regulate growth and survival of plaque-derived vascular smooth muscle cells (VSMC) and influence plaque fate. Recent evidence suggests that accumulation of low density lipoproteins (LDL) in VSMC during the progression of atherogenesis is linked to local changes in IGF-I signaling. We investigated the effects of LDL on the biological actions and downstream signaling pathways mediated by this growth factor in A10 VSMC. METHODS AND RESULTS: We first characterized the effects of LDL on the proliferative and anti-apoptotic actions of IGF-I in A10 VSMC. Native LDL were mitogenic and synergistically enhanced DNA synthesis induced by IGF-I from 4-, 9- up to 7.8-fold, while having no effect on its anti-apoptotic actions. In contrast, oxidized LDL, at oxidation levels that did not modify these actions by themselves, significantly reduced the mitogenic and survival effects of IGF-I by 40% and 60%, respectively. These observations correlated with opposite changes exerted by native and oxidized LDL on the insulin receptor substrate-1 (IRS)-associated PI3 kinase/Akt response to IGF-I. The extracellular signal-regulated kinase (ERK) signaling response was not affected. CONCLUSIONS: Our study demonstrates a previously unidentified modulation of the actions of IGF-I on A10 VSMC by LDL, dependent on their extent of oxidative modification. Our findings suggest that the differential modulation of the PI3 kinase/Akt response to IGF-I play a pivotal role.

Cooperation between low density lipoproteins and IGF-I in the promotion of mitogenesis in vascular smooth muscle cells.

Low density lipoproteins (LDL) are an independent risk factor for atherosclerosis and show synergism with some growth factors in vascular smooth muscle cell (VSMC) proliferation. IGF-I has mitogenic actions on VSMC, which, in turn, show enhanced expression of IGF-I and its receptor when exposed to hypercholesterolemic diets in vivo. To investigate the molecular basis of a possible interaction between LDL and the IGF-I signaling system in VSMC, we used A10 cells, where synergism between both factors in DNA synthesis was demonstrated. IGF-I activates phosphatidylinositol 3-kinase (PI3 kinase) and extracellular signal-regulated MAPK pathways in A10 cells, although insulin receptor substrate-1 (IRS-1)-associated PI3 kinase is more closely linked to IGF-I induced proliferation. LDL, in pathophysiological concentrations, affect the IGF-I signaling pathway at multiple levels: 1) they induce phosphorylation of IGF-I receptor beta and IRS-1 in a time- and dose-dependent manner; 2) they up-regulate IRS-1-associated PI3 kinase/Akt activation in response to IGF-I at early times; and 3) they show additive effects with IGF-I on extracellular signal-regulated MAPK 1/2 phosphorylation. These actions are not present in very low density lipoprotein treatments. Taken together, these results indicate specific cooperation between LDL and the IGF-I signaling pathways and may represent a more general mechanism through which proatherogenic lipoproteins modulate VSMC response to growth factors.

Molecular cloning and characterization of human endothelial nitric oxide synthase.

The constitutive calcium/calmodulin-dependent nitric oxide (NO) synthase expressed in vascular endothelium shares common biochemical and pharmacologic properties with neuronal NO synthase. However, recent cloning and molecular characterization of NO synthase from bovine endothelial cells indicated the existence of a family of constitutive NO synthases. Accordingly, we undertook molecular cloning and sequence analysis of human endothelial NO synthase. Complementary DNA clones predict a protein of 1,203 amino acids sharing 94% identity with the bovine endothelial protein, but only 60% identity with the rat brain NO synthase isoform. Northern blot analysis with an endothelial-derived cDNA identified a 4.6-4.8 kb mRNA transcript in HUVEC and in situ hybridization localized transcripts to vascular endothelium but not neuronal tissue.

Presence of nitric oxide synthase activity in roots and nodules of Lupinus albus.

NO is a widespread messenger molecule in physiology. We were interested in investigating whether an NO-generating system could be present in plants. NO and L-[14C]citrulline were synthesized by roots and nodules of Lupinus albus in an L-arginine-dependent manner. L-[14C]Citrulline production was inhibited by N(G)-monomethyl-L-arginine, a nitric oxide synthase antagonist, in a competitive way. NADPH-diaphorase activity was localized in the vascular bundles in root and nodules, and also in the nodule infected zone. This staining was significantly reduced in the presence of N(G)-monomethyl-L-arginine. These results indicate the presence of a putative nitric oxide synthase in plants.

Regulation of cyclooxygenase-2 expression by nitric oxide in cells.

Two different cyclooxygenases (COXs) are functional in mammals: COX-1 and COX-2. COX-2 is mainly an inducible isoform that shares significant features with inducible nitric oxide synthase (iNOS) in terms of its tissue distribution and participation in pathophysiological phenomena. Furthermore, the product of iNOS catalysis, nitric oxide (NO), is an important regulator of COX-2 activity and expression, and the products of COX-1 and COX-2 (diverse prostanoids) may also influence iNOS expression. Both positive and negative effects of NO on COX-2 expression have been encountered in experimental systems, showing that the outcome of the NO-COX-2 interaction is exquisitely dependent upon the temporal frame and the cell type studied. The pathophysiological significance of NO-COX cross-talk also arises from in vivo studies, in which most evidence points to a positive effect of NO on COX-2 activity and/or expression. This emphasizes the need to understand the underlying mechanisms. Among these, the capacity of NO and its effector cyclic GMP to modulate the function of several target proteins, including transcription factors such as nuclear factor-kappaB and activator protein-1, appears as the key pathway by which NO may regulate COX-2 expression. Given the capacity of some prostanoids to modulate the inflammatory response, the interplay between NO synthase and COX pathways stands at the center of the pathophysiological basis of inflammatory diseases.

Role of reactive oxygen species in the signalling cascade of cyclosporine A-mediated up-regulation of eNOS in vascular endothelial cells.

1. Cyclosporine A (CsA) increases eNOS mRNA expression in bovine cultured aortic endothelial cells (BAEC). As some effects of CsA may be mediated by reactive oxygen species (ROS), present experiments were devoted to test the hypothesis that the CsA-induced eNOS up-regulation could be dependent on an increased synthesis of ROS. 2. CsA induced a dose-dependent increase of ROS synthesis, with the two fluorescent probes used, DHR123 (CsA 1 microM: 305+/-7% over control) and H2DCFDA (CsA 1 microM: 178+/-6% over control). 3. Two ROS generating systems, xanthine plus xanthine oxidase (XXO) and glucose oxidase (GO), increased the expression of eNOS mRNA in BAEC, an effect which was maximal after 8 h of incubation (XXO: 168+/-21% of control values. GO: 208+/-18% of control values). The ROS-dependent increased eNOS mRNA expression was followed by an increase in eNOS activity. 4. The effect of CsA on eNOS mRNA expression was abrogated by catalase, and superoxide dismutase (SOD). In contrast, the antioxidant PDTC augmented eNOS mRNA expression, both in basal conditions and in the presence of CsA. 5. The potential participation of the transcription factor AP-1 was explored. Electrophoretic mobility shift assays were consistent with an increase in AP-1 DNA-binding activity in BAEC treated with CsA or glucose oxidase. 6. The present results support a role for ROS, particularly superoxide anion and hydrogen peroxide, as mediators of the CsA-induced eNOS mRNA up-regulation. Furthermore, they situate ROS as potential regulators of gene expression in endothelial cells, both in physiological and pathophysiological situations.

CsA and FK506 up-regulate eNOS expression: role of reactive oxygen species and AP-1.

Cyclosporine A (CsA) and FK506 increase endothelial nitric oxide synthase (eNOS) mRNA expression in cultured bovine aortic endothelial cells (BAEC). CsA appears to increase eNOS mRNA levels mainly by increasing the rate of transcription, although a small contribution of mRNA stabilization could not be ruled out. CsA and FK506 induced an increase of ROS synthesis with the fluorescent probe used, DHR123. The ROS generating system glucose oxidase (GO) increased the expression of eNOS mRNA in BAEC. This upregulation of eNOS mRNA by CsA or GO was abrogated by catalase. As AP-1 is a redox-sensitive transcription factor and the bovine eNOS promoter has an AP-1 consensus sequence, a role of this factor in the up-regulation of eNOS mRNA was studied. Electrophoretic mobility shift assays were consistent with an increase in AP-1 DNA-binding activity in BAEC treated with CsA or glucose oxidase. The potential participation of ROS and the transcription factor AP-1 in the regulation of eNOS gene expression is suggested.

Nitric oxide as a regulator of gene expression: studies with the transcription factor proteins cJun and p50.

Both oxidative and nitrosative stresses may result in the inactivation of the binding to DNA of redox-sensitive transcription factors. The underlying biochemical mechanisms may involve oxidation or nitrosylation of critical thiols within the DNA binding domains of these proteins. However, S-glutahionylation, the formation of a mixed disulfide between glutathione and the redox-sensitive cysteine residues, has been shown to occur under NO exposure and pro-oxidative conditions in c-Jun, one of the AP-1 constituents. This modification may be functionally important as it is reversible and has been detected in other transcription factors, such as NF-kappaB, by using covalent chromatography with a modified S-nitrosoglutathione sepharose.

Dipyridamole thallium-201 perfusion imaging for the study of ischemic heart disease in hemodialysis patients.

To assess the usefulness of dipyridamole thallium perfusion imaging in the evaluation of myocardial perfusion in hemodialysis (HD), we studied 29 HD patients divided into three groups: A) 13 patients with clinical angina, B) 8 patients without angina but similar in age, sex, time on HD and hematocrit and C) 8 young asymptomatic patients (mean age 33 +/- 9.7 years). Dipyridamole thallium-201 (Tl-201) perfusion imaging revealed myocardial perfusion defects in 8 patients (61%) from group A, 4 (50%) from group B and 1 (12.5%) from group C. These defects were localized in the inferior, posterior and septal segments of the left ventricle. Abnormal myocardial perfusion was associated with age over 50 years and aortic calcifications (p less than 0.05). Eight patients died within the following four years. All had aortic calcifications (p less than 0.001). Our results show that myocardial perfusion defects are frequent even in non-symptomatic HD patients. This suggests that ischemic heart disease could be more frequent than estimated by clinical symptoms alone. Tl-201 scintigraphy may be a useful non-invasive procedure in cardiological evaluation of HD patients.

Variance analysis by use of a low cost desk top calculator.

A simple program for an HP-97 desk top calculator, which can be adapted to an HP-67, is presented. This program detects the presence of an added component of variance in any series classified with a unique criterion. Each series can be formed by any number of data. The program supplies additional information about this component. A brief theoretical description and a practical example are also included.

Effect of Anodal Transcranial Direct Current Stimulation over the Motor Cortex for Cognition

Transcranial direct current stimulation (tDCS) is a non-invasive brain stimulation technique that modulates cortical excitability and influences cognition. The role of the primary motor cortex (M1) in cognition is controversial. Here, we investigated the offline effects of anodal and sham tDCS over M1 on cognitive tasks that require comparable motor skills, but different levels of working memory and attention. Twenty healthy young female adults received anodal tDCS and sham tDCS to the M1 on two separate testing days in a counter balanced order. The cognitive functions outcome variables were the response time from the Attention Switching Task (AST) and Motor Screening Task (MST) tests using the Cambridge Neuropsychological Test Automated Battery before and after the anodal/sham tDCS. Anodal tDCS significantly improved AST response times from baseline in congruent and incongruent condition and MST mean correct latency (all p < 0.05). There was a significant difference for AST tasks variable include AST Switching cost (mean, correct), AST Mean correct latency, in congruent, incongruent, blocks 3, 5 (non-switching blocks), block 7 (switching block) (p < 0.01) and MST mean latency (p < 0.05) between anodal and sham conditions. These results indicate that tDCS is a promising tool to an improvement in response time in task related attention and motor speed. However, this study warrants further research to determine the long-term effect on other cognitive functions and in different age and gender groups.