Search

Neutrophil-endothelial adhesion is a crucial step in vascular inflammation and is recognized as a direct cause of serious atherosclerosis-mediated diseases. We previously demonstrated that high concentrations of glucose increased adhesion in a protein kinase C (PKC)-dependent manner within 48 h of administration by increasing the surface expression of endothelial adhesion molecules. In this study, we focused on the effects of histamine 2 receptor antagonists on endothelial-neutrophil adhesion and on the surface expression of endothelial adhesion molecules mediated by high glucose levels. Histamine 2 receptor antagonists have pleiotropic effects; they not only block the secretion of gastric acid, but also inhibit cell-cell adhesion, resulting in inhibition of metastasis. However, relevant mechanisms of action are not yet fully understood. Of three histamine 2 receptor antagonists (cimetidine, ranitidine, and famotidine), only cimetidine significantly attenuated adhesion mediated by 48-h incubation with 27.8 mM glucose. Cimetidine was found to decrease the surface expression of endothelial adhesion molecules intercellular adhesion molecule-1 and P-selectin, but not E-selectin. To determine the effects of cimetidine on intracellular level, we examined the effects of cimetidine on PKC-induced changes in adhesion, as well as the effects of nitric oxide (NO) synthase inhibitors on cimetidine. We found that NO synthase inhibitors reduced the inhibitory effects of cimetidine, whereas cimetidine did not affect adhesion mediated by a PKC activator. These data suggest that cimetidine acts directly on endothelial cells to inhibit high-glucose-induced expression of adhesion molecules and neutrophil adhesion mediated by increasing endothelial NO production, but not by inhibiting PKC.

Objective: Endothelial-neutrophil adhesion is crucial for vascular injury, the major cause of diabetic vascular complications. On the other hand, platelet aggregation inhibitors, frequently used for diabetic patients with intermittent claudication, have been shown to decrease the incidence of atherosclerosis-mediated diseases (acute myocardial infarction and stroke). However, whether these agents act directly on the endothelial reactions to hyperglycemia remains unclear. Therefore, we examined their direct effects on endothelial-neutrophil adhesion and expression of endothelial adhesion molecules induced by high glucose., Methods and Results: After human endothelial cells were cultured in high glucose medium, neutrophils from healthy volunteers were added and allowed to adhere for 30 min. Adhered neutrophils were quantified by measuring their myeloperoxidase (MPO) activities, and surface expression of endothelial adhesion molecules was determined with an enzyme immunoassay. Of the platelet aggregation inhibitors tested, only cilostazol significantly attenuated the adhesion through decreasing expression of intercellular adhesion molecule-1 (ICAM-1) and P-selectin. In addition, nitric oxide (NO) synthase inhibitors reduced the inhibitory effects of cilostazol, but a protein kinase C (PKC) activator did not., Conclusions: Cilostazol may act directly on endothelial cells to inhibit expression of adhesion molecules and neutrophil adhesion induced by high glucose through increasing NO production.

Background and Aim: Many lines of evidence indicate that hyperinsulinemia might be associated with coronary atherosclerosis, and, currently, there are no effective strategies for preventing this. We previously reported that high insulin enhances neutrophil-transendothelial migration, a process that involves increased surface presentation of platelet endothelial cell adhesion molecule-1 (PECAM-1) through a mitogen-activated protein (MAP) kinase-dependent event. In this current study, we examined if antidiabetic agents, especially K(ATP) channel blockers, might similarly protect against the leukocyte-endothelial cell interactions enhanced by high insulin., Methods: Neutrophils transmigration across umbilical vein endothelial cells (in high insulin medium) with or without K(ATP) channel blockers was performed. Neutrophil migration was quantified by measuring myeloperoxidase, and surface expression of endothelial PECAM-1 was examined using cell-surface enzyme immunoassay., Results: Neutrophil-transendothelial migration and PECAM-1 expression were enhanced by insulin (100 micro U/mL, 24 h) and were attenuated by gliclazide (20 micro M), but not by other K(ATP) channel blockers (glibenclamide, nateglinide, and glimepiride). Neutrophil migration and PECAM-1 expression were also increased by the mitogen-activated protein (MAP) kinase activator, anisomycin (1 micro M), and also attenuated by gliclazide. Nitric oxide (NO) synthase inhibitors did not modify either gliclazide effect., Conclusions: Our results suggest that the K(ATP) channel blocker, gliclazide, blocks high insulin-mediated neutrophil migration and PECAM-1 expression. These gliclazide effects may be mediated through the inhibition of MAP kinase activation and are unrelated to NO production., (Copyright 2004 John Wiley & Sons, Ltd.)

Background and Aim: There are many lines of evidence indicating that hyperinsulinemia but not hyperglycemia is linked to the development of atherosclerotic diseases such as coronary events in diabetic patients. K(ATP) channel blockers of the sulphonylurea class are used widely to treat type 2 diabetes mellitus even with hyperinsulinemia. In this study, we determined whether K(ATP) channel blockers can protect against atherosclerotic processes enhanced by hyperinsulinemia, namely leukocyte-endothelial cell interactions. In addition, we characterized the intracellular mechanisms involved in protective actions of the K(ATP) channel blocker(s)., Method: Studies of adhesion between neutrophils and human umbilical vein endothelial cells incubated in insulin-rich medium with or without K(ATP) channel blockers were performed. Adhered neutrophils were quantified by measuring their myeloperoxidase activities, and surface expression of endothelial ICAM-1 was examined using an enzyme immunoassay., Results: Both neutrophil adhesion and ICAM-1 expression enhanced by high insulin (100 microU/ml, 48 h) were attenuated by gliclazide (20 microM), but not by other K(ATP) channel blockers (glibenclamide, nateglinide, and glimepiride). In addition, both neutrophil adhesion and ICAM-1 expression which were increased by a MAP kinase activator, anisomycin (1 microM), or a PKC activator, phorbol 12-myristate 13-acetate (10 nM) were also attenuated by gliclazide. Nitric oxide (NO) synthase inhibitors did not affect these effects of gliclazide., Conclusions: These results suggest that among K(ATP) channel blockers, only gliclazide can act directly on endothelial cells to inhibit neutrophil-endothelial cell adhesion and ICAM-1 expression enhanced by hyperinsulinemia. These effects of gliclazide are mediated through inhibiting activation of MAP kinase and PKC, unrelated to NO production.

Background and Aims: There is growing evidence that hyperinsulinemia is linked to the development of atherosclerosis in patients with diabetes. We demonstrated previously that high insulin exacerbates neutrophil-endothelial cell adhesion and endothelial intercellular adhesion molecule (ICAM)-1 expression through activation of protein kinase C (PKC) and mitogen-activated protein (MAP) kinase. Though 3-hydroxymethyl-3-glutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) have been employed as therapeutic agents in the treatment of dyslipidemia, which is frequently accompanied by diabetes mellitus; it is not known whether statins protect against leukocyte-endothelial interactions, especially in hyperinsulinemia. In this study, we determined which statin(s) could protect against endothelial reactions to high insulin., Methods: Studies of adhesion between neutrophils from healthy volunteers and human umbilical vein endothelial cells incubated in regular insulin-rich medium with or without statins were performed. Adhered neutrophils were quantified by measuring their myeloperoxidase (MPO) activities, and endothelial expression of ICAM-1 was examined using an enzyme immunoassay., Results: Both the increased neutrophil-endothelial cell adhesion and ICAM-1 expression caused by high insulin (100 microU/ml) for 48 h were significantly attenuated by pretreatment with cerivastatin (0.01 microM), but not by fluvastatin (0.5 microM) or pravastatin (0.05 microM). These protective actions of cerivastatin were attenuated by a key intermediate in the cholesterol biosynthesis pathway, mevalonate (400 microM). In addition, cerivastatin attenuated both neutrophil-endothelial cell adhesion and endothelial ICAM-1 expression enhanced by a MAP kinase activator, anisomycin (1 microM) but not by a PKC activator, PMA (10 nM)., Conclusions: These results suggest that through inhibiting MAP kinase but not PKC activation therapy with cerivastatin would be promising strategy for inhibiting neutrophil-endothelial cell adhesion and endothelial ICAM-1 expression enhanced by high insulin, which is closely correlated with atherosclerosis.

Neutrophil-endothelial adhesion is a crucial step in vascular inflammation, which is recognized as the direct cause of atherosclerosis-mediated serious diseases. We demonstrated previously that high glucose increased adhesion in a protein kinase C (PKC)-dependent manner within 48 h through increasing surface expression of endothelial adhesion molecules. On the other hand, statins, used for patients with hypercholesterolemia, have been shown to decrease the incidence of atherosclerosis-mediated diseases, but direct effects of statins on endothelial cells remain unclear. In this study, we examined the effects of these compounds on high glucose-mediated neutrophil-endothelial adhesion with respect to the participation of PKC and nitric oxide (NO). After human endothelial cells were cultured for 48 h in high glucose medium, neutrophils from healthy volunteers were added and allowed to adhere for 30 min. Adhered neutrophils were quantified by measuring their myeloperoxidase activities, and surface expression of endothelial adhesion molecules was determined with an enzyme immunoassay. Both pravastatin (0.05 microM) and fluvastatin (0.5 microM) significantly attenuated the adhesion mediated by 27.8 mM glucose for 48 h through decreasing surface expression of endothelial adhesion molecules (intercellular adhesion molecule-1, P-selectin, and E-selectin). NO synthase inhibitors reduced the inhibitory effects of statins, whereas statins did not affect the adhesion mediated by a PKC activator. These data suggest that statins act directly on endothelial cells to inhibit expression of adhesion molecules and neutrophil adhesion mediated by high glucose through increasing endothelial NO production, but not by inhibiting PKC.

Background: We previously reported that culture of endothelial cells in the presence of high glucose concentrations (27.8 and 55.5 mM) increase neutrophils adhesion because of the increase in endothelial adhesion molecules expression via activation of a protein kinase C (PKC) pathway. The antidiabetic sulfonylurea gliclazide, but not glibenclamide, inhibited these events, but the mechanisms involved were not clarified then. We present hereafter the results of further investigations of that effect with special reference to PKC activation., Methods: Human umbilical vein endothelial cells (HUVEC) were cultured for 48 h in a glucose-rich medium and neutrophils from healthy volunteers were then added and allowed to adhere for 30 min. Adhered neutrophils were quantified by measuring myeloperoxidase (MPO) activities and the surface expression of endothelial adhesion molecules was determined by enzyme immunoassay., Results: Culture in the presence of a high glucose concentration (27.8 mM for 48 h) increased neutrophils-endothelial cells adhesion and the surface expression of intercellular adhesion molecule-1 (ICAM-1), P-selectin, and E-selectin on the endothelial cells. These phenomena were significantly inhibited by gliclazide (20 microM). On the other hand, phorbol 12-myristate 13-acetate (PMA), a PKC activator, had an effect similar to a high glucose concentration and that effect was also inhibited by gliclazide., Conclusions: These data suggest that gliclazide inhibits high glucose-mediated neutrophils-endothelial cells adhesion and expression of endothelial adhesion molecules through inhibition of a PKC pathway.

Aims/hypothesis: There is increasing evidence that hyperinsulinaemia is linked with the development of atherosclerosis in patients with diabetes. However, the mechanisms by which hyperinsulinaemia causes accelerated atherosclerosis, especially with respect to leukocytes transendothelial migration, are poorly understood. We examined whether hyperinsulinaemia directly affects neutrophil transendothelial migration and surface expression of related endothelial adhesion molecules., Methods: Experiments on the transmigration of neutrophils from healthy volunteers and from patients with Type II (non-insulin-dependent) diabetes mellitus across human umbilical vein endothelial cells cultured in insulin-rich medium using cell-culture inserts were carried out. Migrated neutrophils were quantified by measuring their myeloperoxidase activities, and the surface expression of endothelial adhesion molecules was examined using an enzyme immunoassay., Results: High insulin (over 50 microU/ml for 24 h) enhanced neutrophil transendothelial migration in a dose-dependent manner. This was associated with increased expression of platelet endothelial cell adhesion molecule-1 (PECAM-1) but not of intercellular adhesion molecule-1 (ICAM-1), P-selectin or E-selectin. Both phenomena were attenuated by pretreatment with a tyrosine kinase inhibitor, especially a mitogen-activated protein kinase inhibitor, but not by inhibitors of other second messengers. In addition, a mitogen-activated protein kinase activator, anisomycin, by itself enhanced both neutrophil transendothelial migration and PECAM-1 expression within 3 h in a dose-dependent manner. Pretreatment with nitric oxide synthase inhibitors had no effect on these events., Conclusion/interpretation: These results suggest that hyperinsulinaemia could accelerate atherosclerosis by directly enhancing neutrophil transendothelial migration through increasing endothelial PECAM-1 expression via mitogen-activated protein kinase activation.

Background: We have previously reported that endothelial cells cultured in the presence of high concentrations of glucose (27.8 and 55.5 mM) exhibited enhanced neutrophil adhesion through increased expression of endothelial adhesion molecules via the activation of a protein kinase C (PKC)-dependent pathway. We also found that the aldose reductase inhibitor, epalrestat, inhibited these events, but the mechanisms for this inhibition remained unclear. In this study, we further investigated the inhibitory mechanisms of epalrestat with reference to PKC activation and nitric oxide (NO) production., Methods: Human umbilical vein endothelial cells (HUVECs) were cultured for 48 h in glucose-rich medium and neutrophils from healthy volunteers were then added and allowed to adhere for 30 min. Adhered neutrophils were quantified by measuring myeloperoxidase (MPO) activity and surface expression of endothelial adhesion molecules was determined by enzyme immunoassay., Results: Culture in the presence of a high concentration of glucose (27.8 mM for 48 h) increased neutrophil-endothelial cell adhesion and surface expression of intercellular adhesion molecule-1 (ICAM-1), P-selectin, and E-selectin on endothelial cells. These phenomena were significantly inhibited by epalrestat (10 microM), while NO synthase (NOS) inhibitors reduced the inhibitory effects of this compound. In contrast, 10 nM phorbol 12-myristate 13-acetate (PMA), a PKC activator, showed similar effects as high glucose, and these effects were also inhibited by epalrestat., Conclusions: Our data suggested that epalrestat inhibited high glucose-mediated neutrophil-endothelial cell adhesion and expression of endothelial adhesion molecules not only through inhibition of a PKC-dependent pathway, but also through increased endothelial NO production.

Background: Atherosclerosis and vascular inflammation induced by hyperglycemia are important factors in the promotion of diabetic complications. One of the earliest events in the inflammatory process is increased binding of neutrophils to endothelial cells. Since vascular inflammation has been recently reported to be crucial for the onset of atherosclerosis-mediated serious diseases (acute myocardial infarction, stroke), in this study, we examined the effects of high glucose concentrations on endothelial-neutrophil cell adhesion and surface expression of endothelial adhesion molecules. We also evaluated the effects of various antidiabetic medicines on these events., Methods: Human umbilical vein endothelial cells (HUVECs) were first cultured for 48 h in the glucose-rich medium, and neutrophils from healthy volunteers were then added and allowed to adhere for 30 min. Adhered neutrophils were quantified by measuring myeloperoxidase (MPO) activities, and surface expression of endothelial adhesion molecules was determined using an enzyme immunoassay., Results: High glucose concentrations (over 27.8 mM) increased endothelial-neutrophil cell adhesion and expression of endothelial adhesion molecules (intercellular adhesion molecule-1 (ICAM-1), P-selectin, E-selectin). These events were protein kinase C (PKC) dependent, because PKC inhibitors, but not other intracellular second messenger inhibitors, significantly blocked them. Among antidiabetic medicines, a sulfonylurea, gliclazide (but not glibenclamide or glimepiride), and an aldose reductase inhibitor, epalrestat, significantly inhibited these events; however, a new K(ATP)-channel blocker, netegulinide, a biguanide, metformine, or an insulin sensitizer, troglitazone, did not., Conclusions: Our data is consistent with hyperglycemia-mediated vascular inflammation through increases in neutrophil adhesion and expression of endothelial adhesion molecules. These events might lead to the onset of atherosclerosis-mediated serious diseases, but could be inhibited by something perhaps, such as gliclazide and epalrestat.

Aims/hypothesis: The association of insulin resistance and compensatory hyperinsulinaemia with increased coronary events in diabetic patients is poorly understood. There are few publications about the direct atherogenic actions of insulin on the endothelium compared with those on vascular smooth muscle cells. The aim of this study was to elucidate whether high insulin directly affects neutrophil-endothelial cell adhesion and surface expression of endothelial adhesion molecules. We also examined what intracellular mechanisms are involved in these events., Methods: Studies of adhesion between neutrophils from healthy volunteers and human umbilical vein endothelial cells incubated in insulin-rich medium were carried out. Adhered neutrophils were quantified by measuring their myeloperoxidase activities and surface expression of endothelial adhesion molecules was examined using an enzyme immunoassay., Results: High insulin enhanced neutrophil-endothelial cell adhesion with an increase in the expression of intercellular adhesion molecule-1 but not E-selectin or P-selectin. Both phenomena were attenuated by pretreatment with protein kinase C inhibitors and a mitogen activated protein kinase inhibitor., Conclusions/interpretation: These results suggest that hyperinsulinaemia causes vascular injury by directly exacerbating neutrophil-endothelial cell adhesion through increasing endothelial expression of intercellular adhesion molecule-1 via activation of protein kinase and mitogen activated protein kinase pathways.