Role of endothelial nitric oxide synthase in endothelial activation: insights from eNOS knockout endothelial cells

The objective of this study was to determine whether absence of endothelial nitric oxide synthase (eNOS) affects the expression of cell surface adhesion molecules in endothelial cells. Murine lung endothelial cells (MLECs) were prepared by immunomagnetic bead selection from wild-type and eNOS knockout mice. Wild-type cells expressed eNOS, but eNOS knockout cells did not. Expression of neuronal NOS and inducible NOS was not detectable in cells of either genotype. Upon stimulation, confluent wild-type MLECs produced significant amounts of NO compared with N-monomethyl-L-arginine-treated wild-type cells. eNOS knockout and wild-type cells showed no difference in the expression of E-selectin, P-selectin, intracellular adhesion molecule-1, and vascular cell adhesion molecule-1 as measured by flow cytometry on the surface of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31)-positive cells. Both eNOS knockout and wild-type cells displayed the characteristics of resting endothelium. Adhesion studies in a parallel plate laminar flow chamber showed no difference in leukocyte-endothelial cell interactions between the two genotypes. Cytokine treatment induced endothelial cell adhesion molecule expression and increased leukocyte-endothelial cell interactions in both genotypes. We conclude that in resting murine endothelial cells, absence of endothelial production of NO by itself does not initiate endothelial cell activation or promote leukocyte-endothelial cell interactions. We propose that eNOS derived NO does not chronically suppress endothelial cell activation in an autocrine fashion but serves to counterbalance signals that mediate activation. vascular biology; atherosclerosis; mouse models     

C-peptide inhibits leukocyte-endothelium interaction in the microcirculation during acute endothelial dysfunction.

C-peptide is a cleavage product that comes from processing proinsulin to insulin that induces nitric oxide (NO) -mediated vasodilation. NO modulates leukocyte-endothelium interaction. We hypothesized that C-peptide might inhibit leukocyte-endothelium interaction via increased release of endothelial NO. Using intravital microscopy of the rat mesentery, we measured leukocyte-endothelium interactions after administration of C-peptide to the rat. Superfusion of the rat mesentery with either thrombin or L-NAME consistently and significantly increased the number of rolling, adhering, and transmigrated leukocytes. C-peptide significantly attenuated either thrombin- or L-NAME-induced leukocyte-endothelium interactions in rat mesenteric venules. A control scrambled sequence of C-peptide characterized by the same amino acid composition in a randomized sequence failed to inhibit leukocyte-endothelium interactions. These effects of C-peptide were associated with decreased surface expression of the cell adhesion molecules P-selectin and ICAM-1 on the microvascular endothelium. Endothelial nitric oxide synthase (eNOS) mRNA levels were increased in rats injected with C-peptide. This enhanced eNOS expression was associated with a marked increase in basal NO release from the aorta of C-peptide-treated rats. We conclude that C-peptide is a potent inhibitor of leukocyte-endothelium interaction and that this effect is specifically related to inhibition of endothelial cell adhesion molecules via maintenance of NO release from the vascular endothelium.     

Bioactivity of nitrolinoleate: effects on adhesion molecules and CD40–CD40L system

The vascular effects of nitrolinoleate (LNO₂), an endogenous product of linoleic acid (LA) nitration by nitric oxide-derived species and a potential nitrosating agent, were investigated on rat endothelial-leukocyte interactions. Confocal microscopy analysis demonstrated that LNO₂ was capable to deliver free radical nitric oxide (^·NO) into cells, 5 min after its administration to cultured cells, with a peak of liberation at 30 min. THP-1 monocytes incubated with LNO₂ for 5 min presented nitrosation of CD40, leading to its inactivation. Other anti-inflammatory actions of LNO₂ were observed in vivo by intravital microscopy assays. LNO₂ decreased the number of adhered leukocytes in postcapillary venules of the mesentery network. In addition to this, LNO₂ reduced mRNA and protein expression of β2-integrin in circulating leukocytes, as well as VCAM-1 in endothelial cells isolated from postcapillary venules, confirming its antiadhesive effects on both cell types. Moreover, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a nitric oxide scavenger, partially abolished the inhibitory action of LNO₂ on leukocyte-endothelium interaction, suggesting that the antiadhesion effects of LNO₂ involve a dual role in leukocyte adhesion, acting as a nitric oxide donor as well as through nitric oxide-independent mechanisms. In conclusion, LNO₂ inhibited adhesion molecules expression and promoted ^·NO inactivation of the CD40–CD40L system, both important processes of the inflammatory response.     

Hypoxia causes leukocyte adherence to mesenteric venules in nonacclimatized, but not in acclimatized, rats.

Although the effects of ischemia-reperfusion have received considerable attention, few studies have directly evaluated the microcirculatory response to systemic hypoxia. The overall objective of this study was to assess the effect of environmental hypoxia on adhesive interactions of circulating leukocytes with rat mesenteric venules by using intravital microscopy. Experiments were designed to 1) characterize the adhesive interactions of circulating leukocytes to venules during acute hypoxia produced by a reduction in inspired PO(2), 2) evaluate the role of nitric oxide in these adhesive interactions, 3) determine whether the effect of hypoxia on leukocyte adhesive interactions differs between acclimatized and nonacclimatized rats, and 4) assess whether compensatory changes in nitric oxide formation contribute to this difference. The results showed that acute hypoxia promotes leukocyte-endothelial adherence in mesenteric venules of nonacclimatized rats. The mechanism of this response is consistent with depletion of nitric oxide within the microcirculation. In contrast, no leukocyte-endothelial adherence occurred during hypoxia in rats acclimatized to hypobaric hypoxia. The results are consistent with increased nitric oxide formation due to expression of inducible nitric oxide synthase during the acclimatization period. Further studies are needed to establish the cause of nitric oxide depletion during acute hypoxia as well as to define the compensatory responses that attenuate hypoxia-induced leukocyte-endothelial adherence in the microvasculature of acclimatized rats.     

CD31/PECAM-1 is a ligand for alpha v beta 3 integrin involved in adhesion of leukocytes to endothelium

To protect the body efficiently from infectious organisms, leukocytes circulate as nonadherent cells in the blood and lymph, and migrate as adherent cells into tissues. Circulating leukocytes in the blood have first to adhere to and then to cross the endothelial lining. CD31/PECAM- 1 is an adhesion molecule expressed by vascular endothelial cells, platelets, monocytes, neutrophils, and naive T lymphocytes. It is a transmembrane glycoprotein of the immunoglobulin gene superfamily (IgSF), with six Ig-like homology units mediating leukocyte-endothelial interactions. The adhesive interactions mediated by CD31 are complex and include homophilic (CD31-CD31) or heterophilic (CD31-X) contacts. Soluble, recombinant forms of CD31 allowed us to study the heterophilic interactions in leukocyte adhesion assays. We show that the adhesion molecule alpha v beta 3 integrin is a ligand for CD31. The leukocytes revealed adhesion mediated by the second Ig-like domain of CD31, and this binding was inhibited by alpha v beta 3 integrin-specific antibodies. Moreover alpha v beta 3 was precipitated by recombinant CD31 from cell lysates. These data establish a third IgSF-integrin pair of adhesion molecules, CD31-alpha v beta 3 in addition to VCAM-1, MadCAM-1/alpha 4 integrins, and ICAM/beta 2 integrins, which are major components mediating leukocyte-endothelial adhesion. Identification of a further versatile adhesion pair broadens our current understanding of leukocyte-endothelial interactions and may provide the basis for the treatment of inflammatory disorders and metastasis formation.     

PECAM-1 dampens cytokine levels during LPS-induced endotoxemia by regulating leukocyte trafficking

AimsTo investigate the mechanism by which platelet endothelial cell adhesion molecule 1 (PECAM-1/CD31), an immunoglobulin (Ig)-superfamily cell adhesion and signaling receptor, regulates pro-inflammatory cytokine levels. The purpose of the present investigation was to test the hypothesis that PECAM-1 influences circulating cytokine levels by regulating the trafficking of activated, cytokine-producing leukocytes to sites of inflammation.Main methodsPECAM-1^+/+ and PECAM-1^?/? mice were subjected to lipopolysaccharide (LPS)-induced endotoxemia, and systemic cytokine levels were measured by Bioplex multiplex cytokine assays. Flow cytometry was employed to enumerate leukocytes at inflammatory sites and to measure cytokine synthesis in leukocyte sub-populations. Enzyme-linked immunosorbent assay (ELISA) was used to measure cytokine levels in tissue samples and in supernatants of in vitro-stimulated leukocytes.Key findingsWe confirmed earlier reports that mice deficient in PECAM-1 had greater systemic levels of pro-inflammatory cytokines following intraperitoneal (IP) LPS administration. Interestingly, expression of PECAM-1, in mice, had negligible effects on the level of cytokine synthesis by leukocytes stimulated in vitro with LPS and in peritoneal macrophages isolated from LPS-injected mice. There was, however, excessive accumulation of macrophages and neutrophils in the lungs of PECAM-1-deficient, compared with wild-type, mice — an event that correlated with a prolonged increase in lung pro-inflammatory cytokine levels.SignificanceOur results demonstrate that PECAM-1 normally functions to dampen systemic cytokine levels during LPS-induced endotoxemia by diminishing the accumulation of cytokine-producing leukocytes at sites of inflammation, rather than by modulating cytokine synthesis by leukocytes.     

Inhibition of osteocyte apoptosis by fluid flow is mediated by nitric oxide

Bone unloading results in osteocyte apoptosis, which attracts osteoclasts leading to bone loss. Loading of bone drives fluid flow over osteocytes which respond by releasing signaling molecules, like nitric oxide (NO), that inhibit osteocyte apoptosis and alter osteoblast and osteoclast activity thereby preventing bone loss. However, which apoptosis-related genes are modulated by loading is unknown. We studied apoptosis-related gene expression in response to pulsating fluid flow (PFF) in osteocytes, osteoblasts, and fibroblasts, and whether this is mediated by loading-induced NO production. PFF (0.7 ± 0.3 Pa, 5 Hz, 1 h) upregulated Bcl-2 and downregulated caspase-3 expression in osteocytes. l-NAME attenuated this effect. In osteocytes PFF did not affect p53 and c-Jun, but l-NAME upregulated c-Jun expression. In osteoblasts and fibroblasts PFF upregulated c-Jun, but not Bcl-2, caspase-3, and p53 expression. This suggests that PFF inhibits osteocyte apoptosis via alterations in Bcl-2 and caspase-3 gene expression, which is at least partially regulated by NO.     

Transmural gradient of leukocyte-endothelial interaction in the rat gastrointestinal tract

Gastrointestinal injury usually starts in the superficial mucosa. We investigated whether leukocyte-endothelial interactions were greater in the gastrointestinal mucosa than the submucosa and muscularis in control tissue and after upregulation of adhesion molecules with endotoxin and after chemical insult with nonsteroidal anti-inflammatory drugs. Inactin-anesthetized rats were given either endotoxin, flurbiprofen, or nitric oxide (NO)-flurbiprofen, after which ICAM-1 and P-selectin expression was measured with the dual-label antibody technique. Leukocyte-endothelial interactions in the different gastric layers were assessed after endotoxin using intravital microscopy. Endotoxin caused a two- to threefold increase in ICAM-1 expression in the stomach and duodenum. There was, however, a gradient in expression across the gut wall with the level of expression in the superficial mucosa (per g) being only 10-25% of that in the deeper layers in both control and endotoxin-treated animals. Constituitive expression of P-selectin in control animals was barely detectable. Endotoxin caused a modest increase in mucosal P-selectin but a very significant increase in the deeper layers. Flurbiprofen caused a slight upregulation of ICAM-1 in the gastric mucosa and duodenum, whereas NO-flurbiprofen had no affect on expression. Intravital microscopy revealed no adhesion and virtually no leukocyte rolling in the vessels of the gastric mucosa despite endotoxin treatment. There was, however, some adhesion and significant leukocyte rolling in the submucosa and muscularis. Thus the superficial gastric and duodenal mucosal microcirculations have a much lower density of ICAM-1 and P-selectin and less leukocyte-endothelial interactions than occurs in the deeper layers of the gut wall even during stimulated upregulation with endotoxin.     

L-Selectin Crosslinking Induces Integrin-Dependent Adhesion: Evidence for a Signaling Pathway Involving PTK but not PKC

L-selectin mediates the initial contact of leukocytes with the endothelium prior to extravasation. Here we demonstrate that L-selectin engagement can induce rapid and avid integrin-dependent T cell adhesion to recombinant immobilized cell adhesion molecules (CAMs) including ICAM-1, ICAM-3, and VCAM-1, as well as to the extracellular matrix protein fibronectin (FN). L-selectin-induced adhesion to these integrin ligands shares characteristics with CD3 mAb- or phorbol ester-induced adhesion in requiring metabolic energy. tyrosine kinase and ligand-stimulated Ca⁺⁺ channel activity. However, L-selectin-induced adhesion is distinct from that induced by phorbol ester or CD3 crosslinking in being relatively independent of protein kinase C (PKC) activity and actin polymerization. Consistent with the higher levels of L-selectin expression on CD45RA⁺(naive) cells, L-selectin crosslinking induces a greater proportion of naive relative to memory cell binding to CAMs and FN. In contrast, exposure to phorbol ester or CD3 crosslinking is more effective in inducing CD45RO⁺ (memory) cell adhesion. Thus, in addition to its role in leukocyte capture and rolling on the endothelium. L-selectin may contribute to β1 and β2 integrin-dependent binding and arrest.     

Dehydroepiandrosterone protects the microcirculation of muscle flaps from ischemia-reperfusion injury by reducing the expression of adhesion molecules

Adhesion molecules contribute to ischemia-reperfusion injury by increasing the endothelial adhesion and extravasation of leukocytes. Scientific evidence suggests that presurgical treatment with dehydroepiandrosterone may protect the microvasculature against this damage, but the exact mechanism is not known. The purpose of this study was to investigate the effects of presurgical dehydroepiandrosterone treatment on microcirculatory hemodynamic parameters and the expression of adhesion molecules in a rat cremaster muscle flap model. Twenty male rats were randomly assigned to three experimental groups. In group I (n = 5), the muscle flaps did not receive presurgical treatment. In group II (n = 6), propylene glycol (30 mg/kg), the vehicle for dehydroepiandrosterone, was injected intravenously before ischemia was induced. In group III (n = 9), dehydroepiandrosterone (30 mg/kg) was injected intravenously before ischemia was induced. All flaps were subjected to 6 hours of ischemia and 90 minutes of reperfusion. Microcirculatory variables (functional capillary density, red blood cell velocity in the main flap arteriole, and numbers of rolling, sticking, and transmigrating leukocytes), blood levels of three adhesion molecules (L-selectin, Mac-1 integrin, and CD44), and the numbers of leukocytes expressing those molecules were analyzed. Analysis of the microcirculatory parameters revealed that dehydroepiandrosterone treatment before ischemia had significant preservative effects on the red blood cell velocity and functional capillary density 30 and 90 minutes after reperfusion, compared with the control and vehicle-treated groups. Leukocyte-endothelial cell interactions were also affected by dehydroepiandrosterone treatment, as reflected by significant decreases in the numbers of sticking and transmigrating leukocytes 30 and 90 minutes after reperfusion. In dehydroepiandrosterone-treated animals, leukocytes exhibited lower levels of expression of adhesion molecules after the onset of ischemia, compared with the control groups. In this study, intravenous dehydroepiandrosterone administration reduced the activation of leukocytes and improved red blood cell velocity and capillary perfusion in the muscle flap microcirculation during ischemia-reperfusion injury. This protective effect was most likely the result of delayed expression of Mac-1 integrin, L-selectin, and CD44 molecules on leukocytes.     

Role of inducible nitric oxide synthase in leukocyte extravasation in vivo

Several recent studies have suggested that nitric oxide (NO) derived from the inducible isoform of NO synthase (NOS) may act as an endogenous modulator of the inflammatory response by inhibiting adhesion of leukocytes to endothelial cells in vitro. Few studies have addressed specifically the role of iNOS in regulating leukocyte recruitment in vivo in a model of acute inflammation. Thus, the objective of this study was to assess the role of iNOS in modulating neutrophil (PMN) extravasation in an oyster glycogen-induced model of acute peritonitis in rats. Data obtained in the present study demonstrates that injection (IP) of oyster glycogen induces massive and selective PMN recruitment into the peritoneal cavity of rats at 6 hrs following OG administration. These extravasated cells were found to contain significant amounts of iNOS protein as assessed by Western blot analysis. Treatment of rats with the selective iNOS inhibitor L-iminoethyl-lysine (L-NIL) dramatically reduced NO levels in lavage fluid as measured by decreases in nitrate and nitrite concentrations without significantly affecting iNOS protein levels. Although L-NIL inhibited NO production by >70%, it did not alter oyster glycogen-induced PMN recruitment when compared to vehicle-treated rats. We conclude that PMN-associated, iNOS-derived NO does not play an important role in modulating extravasation of these leukocytes in this model of acute inflammation.     

Inverse association between endogenous glucocorticoid secretion and L-selectin (CD62L) expression in trauma patients

Exogenously administered glucocorticoids downregulate inflammatory host response, i.e. by inhibition of adhesion molecule expression on leukocyte surfaces. Here, possible associations between the trauma-induced endogenous secretion of cortisol and the expression of neutrophil adhesion molecules (L-selectin/CD62L, CD 11b, CD54) were studied in humans. Standardized elective hip arthroplasty was investigated as an exemplary condition of acute inflammation. In 20 patients, blood for quantification of cortisol and adrenocorticotropic hormone was obtained at minutes 10, 20, 30, 60, hours 1, 2, 4 and 10 and days 1,3 and 7. Expression of L-selectin/CD62L, CD11b and CD54 on neutrophil surfaces was determined preoperatively, and postoperatively at hours 1, 2, 3, 4, and 10 and at days 1 and 3. Secretion of both, adrenocorticotropic hormone and cortisol was significantly increased between 1-10 hours after onset of tissue injury. Compared to baseline values, CD11b expression was increased at hour 1 and normalized after day 1, whereas L-selectin/CD62L expression, mirroring this pattern was decreased until day 1. Patients with high endogenous glucocorticoid secretion exhibited significantly decreased expression selectively of L-selectin/CD62L. However, we also observed that glucocorticoids do not directly induce L-selectin shedding from neutrophil surfaces in vitro, arguing for more indirect glucocorticoid action on adhesion molecule expression. Together, this study showed that increased endogenous cortisol secretion is associated with lower expression of L-selectin on neutrophil surfaces in humans that is consistent with a downmodulating role of this neuroendocrine stress response in inflammatory leukocyte recruitment.     

Echinocystic acid,isolated from Gleditsia sinensis fruit,protects endothelial progenitor cells from damage caused by oxLDL via the Akt/eNOS pathway

Aims

Our previous studies revealed that echinocystic acid (EA) showed obvious attenuation of atherosclerosis in rabbits fed a high-fat diet. However, the underlying mechanisms remain to be elucidated. Considering the importance of endothelial progenitor cells (EPCs) in atherosclerosis, we hypothesise that EPCs may be one of the targets for the anti-atherosclerotic potential of EA.

Main methods

After in vitro cultivation, EPCs were exposed to 100 μg/mL of oxidised low-density lipoprotein (oxLDL) and incubated with or without EA (5 and 20 μM) for 48 h. An additional two groups of EPCs (oxLDL + 20 μM EA) were pre-treated with either wortmannin, an inhibitor of the phosphoinositide 3-kinase (PI3K) pathway, or nitro-l-arginine methyl ester (l-NAME), an endothelial nitric oxide synthase (eNOS)-specific inhibitor. Assessment of EPC apoptosis, adhesion, migration, and nitric oxide (NO) release was performed using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) staining, cell counting, caspase-3 activity assay, transwell chamber assay, and Griess reagent, respectively. The protein expression of protein kinase B (Akt) and eNOS was detected using Western blot.

Key findings

Treatment of EPCs with oxLDL induced significant apoptosis and impaired adhesion, migration, and NO production. The deleterious effects of oxLDL on EPCs were attenuated by EA. However, when EPCs were pre-treated with wortmannin or l-NAME, the effects of EA were abrogated. Additionally, oxLDL significantly down-regulated eNOS protein expression as well as repression of eNOS and Akt phosphorylation.

Significance

The inhibitory effect of oxLDL on Akt/eNOS phosphorylation was attenuated by EA. Taken together, the results indicate that EA protects EPCs from damage caused by oxLDL via the Akt/eNOS pathway.     

Interactions of the gastrotropic bacterium Helicobacter pylori with the leukocyte-endothelium adhesion molecules,the selectins--a preliminary report

The deleterious effects of Helicobacter pylori infection of the stomach are largely the result of a vigorous chronic inflammatory response, and include chronic gastritis, peptic ulceration and gastric cancer. We are exploring the possibility that carbohydrate components on H. pylori contribute to the persistent inflammation through interactions with leukocyte-endothelial adhesion molecules of the host. Lipopolysaccharides of most H. pylori strains contain sequences related to the Lewis (Le(x) or Le(a)) antigens. Carbohydrate sequences of this family encompass ligands for the leukocyte-endothelium adhesion molecules of the host, namely, the E- and P-selectins, which are expressed on inflamed endothelia, and L-selectin, which is constitutively expressed on leukocytes. Here we investigate H. pylori isolates from patients with chronic gastritis, duodenal ulcer and gastric cancer for their interactions with the selectins. Our results provide unequivocal evidence of interactions of isolates from each of the diagnostic groups with E- and L-selectins.     

Resistance to renal damage by chronic nitric oxide synthase inhibition in the Wistar-Furth rat

Chronic nitric oxide synthase inhibition (NOSI) causes chronic kidney disease (CKD) in the Sprague Dawley (SD) rat. We previously showed that the Wistar-Furth (WF) rats are resistant to several models of CKD and maintain renal nitric oxide (NO) production compared with SD rats, whereas low-dose NOSI caused progression of CKD in WF rats. Here, we evaluate the impact of high-dose chronic NOSI in WF and SD rats, as well as intrarenal responses to an acute pressor dose of NOSI in the normal WF. Rats were given N(G)-nitro-l-arginine methyl ester (l-NAME) (150 and 300 mg/l for 6-10 wk) in the drinking water after an initial bolus tail vein injection. Both strains showed significant reductions in total NO production with chronic l-NAME. SD given 150 mg/l l-NAME for 6 wk developed proteinuria and renal injury, whereas WF rats receiving 150 mg/l l-NAME for 6-10 wk or 300 mg/l for 6 wk developed no proteinuria and minimal renal injury. Blood pressure was significantly elevated with chronic NOSI in both strains but was higher in the SD rat. There was little impact on renal nitric oxide synthase expression with l-NAME, except that cortical endothelial nitric oxide synthase abundance increased in WF after 6 wk (150 mg/l). Micropuncture experiments with acute pressor NOSI resulted in similar increases in systemic blood pressure in SD and WF rats, whereas WF rats showed a much smaller increment in glomerular blood pressure compared with SD rats. In conclusion, WF rats do not develop renal injury after chronic NOSI at, or above, a dose that causes significant injury in the SD rat. This protection may be associated with protection from glomerular hypertension.     

Involvement of nitric oxide in insulin induced memory improvement

Although brain was considered as an insulin-insensitive organ, recently it has appeared that insulin has some interesting effects on some brain regions like hippocampus. It has been known that intra-hippocampally administered insulin can improve learning and memory. Knowing that insulin can stimulate nitric oxide (NO) synthesis via eNOS activation and also that NO synthase (NOS) inhibitors can affect learning and memory, the aim of this study was to assess if NO is involved in insulin induced memory improvement. Wistar male rats were intra-CA1 cannulated and the effect of post-training and pre-probe trial intra-hippocampal administration of N-nitro-l-arginine methyl ester (l-NAME) (5, 10, 30 μg), insulin + l-NAME ± l-arginine were assessed in a single-day testing version of Morris water maze (MWM) task. Our results show that, l-NAME can prevent insulin induced memory improvement. This drug had no effect on escape latency of a non-spatial visual discrimination task. Therefore, it seems that endogenous nitric oxide has a role in spatial learning and memory improvement caused by insulin.     

Local oxidative and nitrosative stress increases in the microcirculation during leukocytes-endothelial cell interactions

Leukocyte-endothelial cell interactions and leukocyte activation are important factors for vascular diseases including nephropathy, retinopathy and angiopathy. In addition, endothelial cell dysfunction is reported in vascular disease condition. Endothelial dysfunction is characterized by increased superoxide (O(2) (?-)) production from endothelium and reduction in NO bioavailability. Experimental studies have suggested a possible role for leukocyte-endothelial cell interaction in the vessel NO and peroxynitrite levels and their role in vascular disorders in the arterial side of microcirculation. However, anti-adhesion therapies for preventing leukocyte-endothelial cell interaction related vascular disorders showed limited success. The endothelial dysfunction related changes in vessel NO and peroxynitrite levels, leukocyte-endothelial cell interaction and leukocyte activation are not completely understood in vascular disorders. The objective of this study was to investigate the role of endothelial dysfunction extent, leukocyte-endothelial interaction, leukocyte activation and superoxide dismutase therapy on the transport and interactions of NO, O(2)(?-) and peroxynitrite in the microcirculation. We developed a biotransport model of NO, O(2)(?-) and peroxynitrite in the arteriolar microcirculation and incorporated leukocytes-endothelial cell interactions. The concentration profiles of NO, O(2)(?-) and peroxynitrite within blood vessel and leukocytes are presented at multiple levels of endothelial oxidative stress with leukocyte activation and increased superoxide dismutase accounted for in certain cases. The results showed that the maximum concentrations of NO decreased ~0.6 fold, O(2)(?-) increased ~27 fold and peroxynitrite increased ~30 fold in the endothelial and smooth muscle region in severe oxidative stress condition as compared to that of normal physiologic conditions. The results show that the onset of endothelial oxidative stress can cause an increase in O(2)(?-) and peroxynitrite concentration in the lumen. The increased O(2) (?-) and peroxynitrite can cause leukocytes priming through peroxynitrite and leukocytes activation through secondary stimuli of O(2)(?-) in bloodstream without endothelial interaction. This finding supports that leukocyte rolling/adhesion and activation are independent events.     

Expression of adhesion molecules during cadmium hepatotoxicity

Inflammatory processes play a major role in the secondary injury of the liver produced by cadmium (Cd), and infiltration of neutrophils at the site of necrosis is a common observation. Although the infiltration of leukocytes (mainly neutrophils) into sites of injuried tissue within liver during Cd toxicity is mediated by adhesion molecules, little is known about expression of these adhesion molecules during Cd hepatotoxicity. In the present study, the expression of E-, P-selectin, intracellular adhesion molecule-1 (ICAM-1) and platelet-endothelial adhesion molecule-1 (PECAM-1) was analyzed by immunohistochemistry and immunofluoresence during Cd-induced hepatotoxicity in male rats. In contrast to E-, and P-selectin, ICAM-1 and PECAM-1 were constitutively expressed on sinusoidal endothelial cells of control liver. However, P-selectin was not induced within the liver by Cd administration, whereas E-selectin expression was induced in the liver with a marked increase in immunostaining on sinusoidal endothelial cells from 12 h to 7 days. Also, there was an upregulation in ICAM-1 immunostaining on sinusoidal endothelial cells from 12 h to 7 days after Cd administration, whereas there was no obvious change of PECAM-1 immunostaining on sinusoidal endothelial cells until 24 h. However, PECAM-1 expression was markedly decreased at 48 h but significantly increased at 7 days after Cd administration compared to control liver. Taken together, upregulation of E-selectin and ICAM-1 with biphasic changes in PECAM-1 expression within liver after Cd administration suggests an important role for these adhesion molecules during Cd hepatoxicity.     

Nitric oxide inhibits metamorphosis in larvae of Crepidula fornicata, the slippershell snail

This paper concerns the role of nitric oxide (NO) in controlling metamorphosis in the marine gastropod Crepidula fornicata. Metamorphosis was stimulated by the nitric oxide synthase (NOS) inhibitors AGH (aminoguanidine hemisulfate) and SMIS (S-methylisothiourea sulfate) at concentrations of about 100-1000 micromol l(-1) and 50-200 micromol l(-1), respectively. Metamorphosis was not, however, induced by the NOS inhibitor l-NAME (l-N(G)-nitroarginine methyl ester) at even the highest concentration tested, 500 micromol l(-1). Moreover, pre-incubation with l-NAME at 20 and 80 micromol l(-1) did not increase the sensitivity of competent larvae to excess K(+), a potent inducer of metamorphosis in this species; we suggest that either l-NAME is ineffective in suppressing NO production in larvae of C. fornicata, or that it works only on the constitutive isoform of the enzyme. In contrast, metamorphosis was potentiated by the guanylate cyclase inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3, -a]quinoxalin-1-one) in response to a natural metamorphic inducer derived from conspecific adults. Because NO typically stimulates cGMP production through the activation of soluble guanylate cyclase, this result supports the hypothesis that NO acts as an endogenous inhibitor of metamorphosis in C. fornicata. The expression of NOS, shown by immunohistochemical techniques, was detected in the apical ganglion of young larvae but not in older larvae, further supporting the hypothesis that metamorphosis in C. fornicata is made possible by declines in the endogenous concentration of NO during development.