Nonspecific blockade of vascular free radical signals by methylated arginine analogues

Methylated arginine analogues are often used as probes of the effect of nitric oxide; however, their specificity is unclear and seems to be frequently overestimated. This study analyzed the effects of NG-methyl-L-arginine (L-NMMA) on the endothelium-dependent release of vascular superoxide radicals triggered by increased flow. Plasma ascorbyl radical signals measured by direct electron paramagnetic resonance spectroscopy in 25 rabbits increased by 3.8 ± 0.7 nmol/l vs baseline (28.7 ± 1.4 nmol/l, P<0.001) in response to papaverine-induced flow increases of 121 ± 12%. In contrast, after similar papaverine-induced flow increases simultaneously with L-NMMA infusions, ascorbyl levels were not significantly changed compared to baseline. Similar results were obtained in isolated rabbit aortas perfused ex vivo with the spin trap a-phenyl-N-tert-butylnitrone (N = 22). However, in both preparations, this complete blockade was not reversed by co-infusion of excess L-arginine and was also obtained by N-methyl-D-arginine, thus indicating that it is not related to nitric oxide synthase. L-arginine alone was ineffective, as previously demonstrated for NG-methyl-L-arginine ester (L-NAME). In vitro, neither L-arginine nor its analogues scavenged superoxide radicals. This nonspecific activity of methylated arginine analogues underscores the need for careful controls in order to assess nitric oxide effects, particularly those related to interactions with active oxygen species.

Integrins in vascular development

Many growth factors and their protein kinase receptors play a role in regulating vascular development. In addition, cell adhesion molecules, such as integrins and their ligands in the extracellular matrix, play important roles in the adhesion, migration, proliferation, survival and differentiation of the cells that form the vasculature. Some integrins are known to be regulated by angiogenic growth factors and studies with inhibitors of integrin functions and using strains of mice lacking specific integrins clearly implicate some of these molecules in vasculogenesis and angiogenesis. However, the data are incomplete and sometimes discordant and it is unclear how angiogenic growth factors and integrin-mediated adhesive events cooperate in the diverse cell biological processes involved in forming the vasculature. Consideration of the results suggests working hypotheses and raises questions for future research directions.

Photodynamic DNA damage induced by phycocyanin and its repair in Saccharomyces cerevisiae

In the present study, we analyzed DNA damage induced by phycocyanin (PHY) in the presence of visible light (VL) using a set of repair endonucleases purified from Escherichia coli. We demonstrated that the profile of DNA damage induced by PHY is clearly different from that induced by molecules that exert deleterious effects on DNA involving solely singlet oxygen as reactive species. Most of PHY-induced lesions are single strand breaks and, to a lesser extent, base oxidized sites, which are recognized by Nth, Nfo and Fpg enzymes. High pressure liquid chromatography coupled to electrochemical detection revealed that PHY photosensitization did not induce 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) at detectable levels. DNA repair after PHY photosensitization was also investigated. Plasmid DNA damaged by PHY photosensitization was used to transform a series of Saccharomyces cerevisiae DNA repair mutants. The results revealed that plasmid survival was greatly reduced in rad14 mutants, while the ogg1 mutation did not modify the plasmid survival when compared to that in the wild type. Furthermore, plasmid survival in the ogg1 rad14 double mutant was not different from that in the rad14 single mutant. The results reported here indicate that lethal lesions induced by PHY plus VL are repaired differently by prokaryotic and eukaryotic cells. Morever, nucleotide excision repair seems to play a major role in the recognition and repair of these lesions in Saccharomyces cerevisiae.

Magnesium chloride alone or in combination with diazepam fails to prevent hippocampal damage following transient forebrain ischemia

In the central nervous system, magnesium ion (Mg2+) acts as an endogenous modulator of N-methyl-D-aspartate (NMDA)-coupled calcium channels, and may play a major role in the pathomechanisms of ischemic brain damage. In the present study, we investigated the effects of magnesium chloride (MgCl2, 2.5, 5.0 or 7.5 mmol/kg), either alone or in combination with diazepam (DZ), on ischemia-induced hippocampal cell death. Male Wistar rats (250-300 g) were subjected to transient forebrain ischemia for 15 min using the 4-vessel occlusion model. MgCl2 was applied systemically (sc) in single (1x, 2 h post-ischemia) or multiple doses (4x, 1, 2, 24 and 48 h post-ischemia). DZ was always given twice, at 1 and 2 h post-ischemia. Thus, ischemia-subjected rats were assigned to one of the following treatments: vehicle (0.1 ml/kg, N = 34), DZ (10 mg/kg, N = 24), MgCl2 (2.5 mmol/kg, N = 10), MgCl2 (5.0 mmol/kg, N = 17), MgCl2 (7.5 mmol/kg, N = 9) or MgCl2 (5 mmol/kg) + DZ (10 mg/kg, N = 14). Seven days after ischemia the brains were analyzed histologically. Fifteen minutes of ischemia caused massive pyramidal cell loss in the subiculum (90.3%) and CA1 (88.4%) sectors of the hippocampus (P<0.0001, vehicle vs sham). Compared to the vehicle-treated group, all pharmacological treatments failed to attenuate the ischemia-induced death of both subiculum (lesion: 86.7-93.4%) and CA1 (lesion: 85.5-91.2%) pyramidal cells (P>0.05). Both DZ alone and DZ + MgCl2 reduced rectal temperature significantly (P<0.05). No animal death was observed after drug treatment. These data indicate that exogenous magnesium, when administered systemically post-ischemia even in different multiple dose schedules, alone or with diazepam, is not useful against the histopathological effects of transient global cerebral ischemia in rats.

Ischemic preconditioning reduces peripheral oxidative damage associated with brain ischemia in rats

Brain ischemia followed by reperfusion causes neuronal death related to oxidative damage. Furthermore, it has been reported that subjects suffering from ischemic cerebrovascular disorders exhibit changes in circulating platelet aggregation, a characteristic that might be important for their clinical outcome. In the present investigation we studied tert-butyl hydroperoxide-initiated plasma chemiluminescence and thiol content as measures of peripheral oxidative damage in naive and preconditioned rats submitted to forebrain ischemia produced by the 4-vessel occlusion method. Rats were submitted to 2 or 10 min of global transient forebrain ischemia followed by 60 min or 1, 2, 5, 10 or 30 days of reperfusion. Preconditioned rats were submitted to a 10-min ischemic episode 1 day after a 2-min ischemic event (2 + 10 min), followed by 60 min or 1 or 2 days of reperfusion. It has been demonstrated that such preconditioning protects against neuronal death in rats and gerbils submitted to a lethal (10 min) ischemic episode. The results show that both 2 and 10 min of ischemia cause an increase of plasma chemiluminescence when compared to control and sham rats. In the 2-min ischemic group, the effect was not present after reperfusion. In the 10-min ischemic group, the increase was present up to 1 day after recirculation and values returned to control levels after 2 days. However, rats preconditioned to ischemia (2 + 10 min) and reperfusion showed no differences in plasma chemiluminescence when compared to controls. We also analyzed plasma thiol content since it has been described that sulfhydryl (SH) groups significantly contribute to the antioxidant capacity of plasma. There was a significant decrease of plasma thiol content after 2, 10 and 2 + 10 min of ischemia followed by reperfusion when compared to controls. We conclude that ischemia may cause, along with brain oxidative damage and cell death, a peripheral oxidative damage that is reduced by the preconditioning phenomenon.

Vascular changes after cardiopulmonary bypass and ischemic cardiac arrest: roles of nitric oxide synthase and cyclooxygenase

Cardiac surgery involving ischemic arrest and extracorporeal circulation is often associated with alterations in vascular reactivity and permeability due to changes in the expression and activity of isoforms of nitric oxide synthase and cyclooxygenase. These inflammatory changes may manifest as systemic hypotension, coronary spasm or contraction, myocardial failure, and dysfunction of the lungs, gut, brain and other organs. In addition, endothelial dysfunction may increase the occurrence of late cardiac events such as graft thrombosis and myocardial infarction. These vascular changes may lead to increased mortality and morbidity and markedly lengthen the time of hospitalization and cost of cardiac surgery. Developing a better understanding of the vascular changes operating through nitric oxide synthase and cyclooxygenase may improve the care and help decrease the cost of cardiovascular operations.

Blockade of the action of nitric oxide in human septic shock increases systemic vascular resistance and has detrimental effects on pulmonary function after a short infusion of methylene blue

To investigate the role of nitric oxide in human sepsis, ten patients with severe septic shock requiring vasoactive drug therapy and mechanical ventilation were enrolled in a prospective, open, non-randomized clinical trial to study the acute effects of methylene blue, an inhibitor of guanylate cyclase. Hemodynamic and metabolic variables were measured before and 20, 40, 60, and 120 min after the start of a 1-h intravenous infusion of 4 mg/kg of methylene blue. Methylene blue administration caused a progressive increase in mean arterial pressure (60 [55-70] to 70 [65-100] mmHg, median [25-75th percentiles]; P<0.05), systemic vascular resistance index (649 [479-1084] to 1066 [585-1356] dyne s-1 cm-5 m-2; P<0.05) and the left ventricular stroke work index (35 [27-47] to 38 [32-56] g m-1 m-2; P<0.05) from baseline to 60 min. The pulmonary vascular resistance index increased from 150 [83-207] to 186 [121-367] dyne s-1 cm-5 m-2 after 20 min (P<0.05). Mixed venous saturation decreased from 65 [56-76] to 63 [55-69]% (P<0.05) after 60 min. The PaO2/FiO2 ratio decreased from 168 [131-215] to 132 [109-156] mmHg (P<0.05) after 40 min. Arterial lactate concentration decreased from 5.1 ± 2.9 to 4.5 ± 2.1 mmol/l, mean ± SD (P<0.05) after 60 min. Heart rate, cardiac filling pressures, cardiac output, oxygen delivery and consumption did not change. Methylene blue administration was safe and no adverse effect was observed. In severe human septic shock, a short infusion of methylene blue increases systemic vascular resistance and may improve myocardial function. Although there was a reduction in blood lactate concentration, this was not explained by an improvement in tissue oxygenation, since overall oxygen availability did not change. However, there was a significant increase in pulmonary vascular tone and a deterioration in gas exchange. Further studies are needed to demonstrate if nitric oxide blockade with methylene blue can be safe for patients with septic shock and, particularly, if it has an effect on pulmonary function.

Ghost protein damage by peroxynitrite and its protection by melatonin

We have studied the effect of peroxynitrite (ONOO-) on the membrane cytoskeleton of red blood cells and its protection by melatonin. Analysis of the protein fraction of the preparation by SDS-PAGE revealed a dose-dependent (0-600 µM ONOO-) disappearance at pH 7.4 of the main proteins: spectrin, band 3, and actin, with the concomitant formation of high-molecular weight aggregates resistant to reduction by ß-mercaptoethanol (2%) at room temperature for 20 min. These aggregates were not solubilized by 8 M urea. Incubation of the membrane cytoskeleton with ONOO- was characterized by a marked depletion of free sulfhydryl groups (50% at 250 µM ONOO-). However, a lack of effect of ß-mercaptoethanol suggests that, under our conditions, aggregate formation is not mediated only by sulfhydryl oxidation. The lack of a protective effect of the metal chelator diethylenetriaminepentaacetic acid confirmed that ONOO--induced oxidative damage does not occur only by a transition metal-dependent mechanism. However, we demonstrated a strong protection against cytoskeletal alterations by desferrioxamine, which has been described as a direct scavenger of the protonated form of peroxynitrite. Desferrioxamine (0.5 mM) also inhibited the loss of tryptophan fluorescence observed when the ghosts were treated with ONOO-. Glutathione, cysteine, and Trolox® (1 mM), but not mannitol (100 mM), were able to protect the proteins against the effect of ONOO- in a dose-dependent manner. Melatonin (0-1 mM) was especially efficient in reducing the loss of spectrin proteins when treated with ONOO- (90% at 500 µM melatonin). Our findings show that the cytoskeleton, and in particular spectrin, is a sensitive target for ONOO-. Specific antioxidants can protect against such alterations, which could seriously impair cell dynamics and generate morphological changes.

Biophysical characteristics of gap junctions in vascular wall cells: implications for vascular biology and disease

The role gap junction channels play in the normal and abnormal functioning of the vascular wall is the subject of much research. The biophysical properties of gap junctions are an essential component in understanding how gap junctions function to allow coordinated relaxation and contraction of vascular smooth muscle. This study reviews the properties thus far elucidated and relates those properties to tissue function. We ask how biophysical and structural properties such as gating, permselectivity, subconductive states and channel type (heteromeric vs homotypic vs heterotypic) might affect vascular smooth muscle tone.

Kallikrein-like amidase activity in renal ischemia and reperfusion

We assessed a kallikrein-like amidase activity probably related to the kallikrein-kinin system, as well as the participation of leukocyte infiltration in renal ischemia and reperfusion. Male C57BL/KSJmdb mice were subjected to 20 or 60 min of ischemia and to different periods of reperfusion. A control group consisted of sham-operated mice, under similar conditions, except for ischemia induction. Kallikrein-like amidase activity, Evans blue extravasation and myeloperoxidase activity were measured in kidney homogenates, previously perfused with 0.9% NaCl. Plasma creatinine concentration increased only in the 60-min ischemic group. After 20 min of ischemia and 1 or 24 h of reperfusion, no change in kallikrein-like amidase activity or Evans blue extravasation was observed. In the mice subjected to 20 min of ischemia, edema was evident at 1 h of reperfusion, but kidney water content returned to basal levels after 24 h of reperfusion. In the 60-min ischemic group, kallikrein-like amidase activity and Evans blue extravasation showed a similar significant increase along reperfusion time. Kallikrein-like amidase activity increased from 4 nmol PNA mg protein-1 min-1 in the basal condition to 15 nmol PNA mg protein-1 min-1 at 10 h of reperfusion. For dye extravasation the concentration measured was near 200 µg of Evans blue/g dry tissue in the basal condition and 1750 µg of Evans blue/g dry tissue at 10 h of reperfusion. No variation could be detected in the control group. A significant increase from 5 to 40 units of DAbs 655 nm g wet tissue-1 min-1 in the activity of the enzyme myeloperoxidase was observed in the 60-min ischemic group, when it was evaluated after 24 h of reperfusion. Histological analysis of the kidneys showed migration of polymorphonuclear leukocytes from the vascular bed to the interstitial tissue in the 60-min ischemic group after 24 h of reperfusion. We conclude that the duration of ischemia is critical for the development of damage during reperfusion and that the increase in renal cortex kallikrein-like amidase activity probably released from both the kidney and leukocytes may be responsible, at least in part, for the observed effects, probably through direct induction of increased vascular permeability.

Angiotensin II-mediated vascular smooth muscle cell growth signaling

The mechanism by which Ang II stimulates the growth of vascular smooth muscle cells was investigated by measuring the phosphorylation of mitogen-activated protein kinases ERK 1 and ERK 2. Ca2+ ionophore was found to have effects practically analogous to Ang II. We found that the signaling pathway involves the activation of epidermal growth factor receptor (EGFR) kinase, activation of the adaptor proteins Shc and Grb2, and the small G-protein Ras. Although the mechanism of AT1- (or Ca2+)-induced activation of EGFR is not yet clear, we have found that calcium-dependent protein kinase CAKß/PYK2 and c-Src are involved in this process. These studies indicate a transactivation mechanism that utilizes EGFR as a bridge between a Gq-coupled receptor and activation of phosphotyrosine generation.

Evaluation of radioinduced damage and repair capacity in blood lymphocytes of breast cancer patients

Genetic damage caused by ionizing radiation and repair capacity of blood lymphocytes from 3 breast cancer patients and 3 healthy donors were investigated using the comet assay. The comets were analyzed by two parameters: comet tail length and visual classification. Blood samples from the donors were irradiated in vitro with a 60Co source at a dose rate of 0.722 Gy/min, with a dose range of 0.2 to 4.0 Gy and analyzed immediately after the procedure and 3 and 24 h later. The basal level of damage and the radioinduced damage were higher in lymphocytes from breast cancer patients than in lymphocytes from healthy donors. The radioinduced damage showed that the two groups had a similar response when analyzed immediately after the irradiations. Therefore, while the healthy donors presented a considerable reduction of damage after 3 h, the patients had a higher residual damage even 24 h after exposure. The repair capacity of blood lymphocytes from the patients was slower than that of lymphocytes from healthy donors. The possible influence of age, disease stage and mutations in the BRCA1 and BRCA2 genes are discussed. Both parameters adopted proved to be sensitive and reproducible: the dose-response curves for DNA migration can be used not only for the analysis of cellular response but also for monitoring therapeutic interventions. Lymphocytes from the breast cancer patients presented an initial radiosensitivity similar to that of healthy subjects but a deficient repair mechanism made them more vulnerable to the genotoxic action of ionizing radiation. However, since lymphocytes from only 3 patients and 3 normal subjects were analyzed in the present paper, additional donors will be necessary for a more accurate evaluation.

Development of new heparin-like compounds and other antithrombotic drugs and their interaction with vascular endothelial cells

The anticlotting and antithrombotic activities of heparin, heparan sulfate, low molecular weight heparins, heparin and heparin-like compounds from various sources used in clinical practice or under development are briefly reviewed. Heparin isolated from shrimp mimics the pharmacological activities of low molecular weight heparins. A heparan sulfate from Artemia franciscana and a dermatan sulfate from tuna fish show a potent heparin cofactor II activity. A heparan sulfate derived from bovine pancreas has a potent antithrombotic activity in an arterial and venous thrombosis model with a negligible activity upon the serine proteases of the coagulation cascade. It is suggested that the antithrombotic activity of heparin and other antithrombotic agents is due at least in part to their action on endothelial cells stimulating the synthesis of an antithrombotic heparan sulfate.

Ambulatory blood pressure and Doppler echocardiographic indexes of borderline hypertensive men presenting an exaggerated blood pressure response during dynamic exercise

Borderline hypertension (BH) has been associated with an exaggerated blood pressure (BP) response during laboratory stressors. However, the incidence of target organ damage in this condition and its relation to BP hyperreactivity is an unsettled issue. Thus, we assessed the Doppler echocardiographic profile of a group of BH men (N = 36) according to office BP measurements with exaggerated BP in the cycloergometric test. A group of normotensive men (NT, N = 36) with a normal BP response during the cycloergometric test was used as control. To assess vascular function and reactivity, all subjects were submitted to the cold pressor test. Before Doppler echocardiography, the BP profile of all subjects was evaluated by 24-h ambulatory BP monitoring. All subjects from the NT group presented normal monitored levels of BP. In contrast, 19 subjects from the original BH group presented normal monitored BP levels and 17 presented elevated monitored BP levels. In the NT group all Doppler echocardiographic indexes were normal. All subjects from the original BH group presented normal left ventricular mass and geometrical pattern. However, in the subjects with elevated monitored BP levels, fractional shortening was greater, isovolumetric relaxation time longer, and early to late flow velocity ratio was reduced in relation to subjects from the original BH group with normal monitored BP levels (P<0.05). These subjects also presented an exaggerated BP response during the cold pressor test. These results support the notion of an integrated pattern of cardiac and vascular adaptation during the development of hypertension.

Effects of lipopolysaccharide on low- and high-density cultured rabbit vascular smooth muscle cells: differential modulation of nitric oxide release, ERK1/ERK2 MAP kinase activity, protein tyrosine phosphatase activity, and DNA synthesis

Previous studies have shown that exogenously generated nitric oxide (NO) inhibits smooth muscle cell proliferation. In the present study, we stimulated rabbit vascular smooth muscle cells (RVSMC) with E. coli lipopolysaccharide (LPS), a known inducer of NO synthase transcription, and established a connection between endogenous NO, phosphorylation/dephosphorylation-mediated signaling pathways, and DNA synthesis. Non-confluent RVSMC were cultured with 0, 5, 10, or 100 ng/ml of the endotoxin. NO release was increased by 86.6% (maximum effect) in low-density cell cultures stimulated with 10 ng/ml LPS as compared to non-stimulated controls. Conversely, LPS (5 to 100 ng/ml) did not lead to enhanced NO production in multilayered (high density) RVSMC. DNA synthesis measured by thymidine incorporation showed that LPS was mitogenic only to non-confluent RVSMC; furthermore, the effect was prevented statistically by aminoguanidine (AG), a potent inhibitor of the inducible NO synthase, and oxyhemoglobin, an NO scavenger. Finally, there was a cell density-dependent LPS effect on protein tyrosine phosphatase (PTP) and ERK1/ERK2 mitogen-activated protein (MAP) kinase activities. Short-term transient stimulation of ERK1/ERK2 MAP kinases was maximal at 12 min in non-confluent RVSMC and was prevented by preincubation with AG, whereas PTP activities were inhibited in these cells after 24-h LPS stimulation. Conversely, no significant LPS-mediated changes in kinase or phosphatase activities were observed in high-density cells. LPS-induced NO generation by RVSMC may switch on a cell density-dependent proliferative signaling cascade, which involves the participation of PTP and the ERK1/ERK2 MAP kinases.