Low-density lipoprotein induced actin cytoskeleton reorganization in endothelial cells: mechanisms of action.

The inhibitory effects of the specific NADPH oxidase inhibitor, apocynin, and non-specific NADPH oxidase inhibitors, nordihydroguaiaretic acid (NDGA) and SKF525A, on the disruption of dense peripheral bands and formation of stress fibers in cultured human umbilical vein endothelial cells exposed to atherogenic low-density lipoprotein (LDL) levels has been investigated. Endothelial cells (EC) in vitro and in vivo exposed to high LDL-cholesterol levels have cytoskeletal remodeling with stress fiber formation and loss of dense peripheral bands. Cultured EC incubated with exogenously applied hydrogen peroxide (H2O2: 1 mM) have cytoskeletal structural changes much similar to those observed with high LDL exposure. Previous studies have 1) demonstrated that exposure to atherogenic LDL levels causes heightened EC H2O2 production, 2) identified the reactive oxygen species source, NADPH oxidase, in EC, and 3) shown that the specific NADPH oxidase inhibitor, apocynin, and non-specific NADPH oxidase inhibitors, NDGA and SKF525A, suppress H2O2 production increases in high LDL-perturbed EC. In the present study, the cytoskeletal structure of EC exposed to 330 mg/dl LDL-cholesterol, and incubated with or without apocynin, NDGA and SKF525A, was examined. Each of these compounds promoted the retention of dense peripheral bands and minimized stress fiber formation. These findings are consistent with NADPH oxidase and it's reactive oxygen species byproducts modulating the cytoskeleton reorganization observed in high LDL-induced EC perturbation.

Endothelial cell oxidant production: effect of NADPH oxidase inhibitors.

The effects of known leukocyte NADPH oxidase inhibitors on general cellular oxidant production in cultured human endothelial cells (EC) has been investigated. EC were stimulated with 10 nM phorbol 12-myristate 13-acetate and cellular oxidant production measured in the presence and absence of inhibitors that act on various substituents of the oxidase complex and its activation pathways. The effects of the cytosolic oxidase subunit translocation inhibitors, catechols (3,4-dihydroxybenzaldehyde, caffeic acid, and protocatechuic acid), ortho-methoxy-substituted catechols (apocynin, vanillin, and 4-nitroguaiacol), and quinone, 1,4-naphthoquinone; flavoprotein inhibitors, diphenylene iodonium and quinacrine; haem ligands, imidazole and pyridine; directly acting thiol reagents, disulfiram and penicillamine; NADPH analogue, Cibacron Blue; redox active inhibitors, quercetin and esculetin; intracellular calcium antagonist, TMB-8; and calmodulin antagonists, W-7 and trifluoperazine, were determined. All compounds reduced oxidant production in stimulated EC. These findings add to previous observations suggesting the presence of a functionally active NADPH oxidase in EC. Identifying the major cellular reactive oxygen species source in perturbed EC will provide new insights into our understanding of endothelial dysfunction, which has been hypothesized to be a major contributing factor in the pathogenesis of atherosclerosis.

Identification of a functional leukocyte-type NADPH oxidase in human endothelial cells :a potential atherogenic source of reactive oxygen species.

Cultured human endothelial cells (EC) exposed to atherogenic low-density lipoprotein levels have increased reactive oxygen species (ROS) generation. The enzyme responsible for this ROS production elevation is unknown. We have examined for the presence of a functional leukocyte-type NADPH oxidase in EC to elucidate whether this enzyme could be the ROS source. The plasma membrane fraction of disrupted EC showed a reduced-minus-oxidized difference spectra with absorption peaks identical to those observed in the spectra of the leukocyte NADPH oxidase component, cytochrome b558. Western-blot analysis, using anti-gp91 -phox. anti -p22-phox. anti -p47-phox. and anti -p67-phox antibodies, demonstrated the protein expression of NADPH oxidase subunits in EC. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed the mRNA expression of gp91-phox, p22-phox, p47-phox, and p67-phox in EC. Sonicates from unstimulated EC produced no measurable superoxide; whereas, exogenously applied arachidonic acid activated superoxide generation in a manner that was dependent upon the presence of NADPH and both membrane and cytosolic fractions combined. Apocynin, a specific leukocyte NADPH oxidase inhibitor, was shown by Western-blot analysis of membrane and cytoplasmic fractions to inhibit the translocation of p47-phox to the membrane of stimulated EC. These findings support the presence of a functionally active leukocyte-type NADPH oxidase in EC. NADPH oxidase could be the major cellular ROS source in EC perturbation, which has been hypothesized to be a major contributing factor in the pathogenesis of atherosclerosis.

Thrombin stimulated reactive oxygen species production in cultured human endothelial cells.

In order to study the major cellular source of reactive oxygen species (ROS) in perturbed human endothelial cells (EC), the effect of thrombin, a phospholipase A2 activator, on cultured EC ROS generation has been investigated. EC were incubated with 0.1-1 unit/ml thrombin and cellular superoxide anion (O(-)2) release and hydrogen peroxide (H2O2) production measured. Thrombin exposure caused an elevation in EC O(-)2 release and H2O2 production. The effects of protein kinase C, arachidonic acid metabolism, NADPH oxidase, and phospholipase A2 inhibitors on thrombin-induced EC H2O2 production were examined. EC were exposed to 0.5 unit/ml thrombin and cellular H2O2 production measured in the presence and absence of the protein kinase C inhibitor, H-7; arachidonic acid metabolism inhibitors, indomethacin, nordihydroguaiaretic acid, and SKF525A; NADPH oxidase inhibitor, apocynin; and phospholipase A2 inhibitor, 4-bromophenacyl bromide. All inhibitors, with the exception of H-7 and indomethacin, suppressed thrombin-induced EC H2O2 production. The pattern of effects of these metabolic antagonists on thrombin-induced EC ROS production is similar to that previously reported on ROS production in EC exposed to high low-density lipoprotein levels, and in stimulated leukocytes. These findings further implicate NADPH oxidase as a major ROS source in EC.

Low-density lipoprotein stimulated peroxide production and endocytosis in cultured human endothelial cells: mechanisms of action.

The effects of arachidonic acid metabolism and NADPH oxidase inhibitor on the hydrogen peroxide (H2O2) generation and endocytotic activity of cultured human endothelial cells (EC) exposed to atherogenic low-density lipoprotein (LDL) levels have been investigated. EC were incubated with 240 mg/dl LDL cholesterol and cellular H2O2 production and endocytotic activity measured in the presence and absence of the arachidonic acid metabolism inhibitors, indomethacin, nordihydroguaiaretic acid, and SKF525A, and NADPH oxidase inhibitor, apocynin. All inhibitors, with the exception of indomethacin, markedly reduced high LDL-induced increases in EC H2O2 generation and endocytotic activity. EC exposed to exogenously applied arachidonic acid had cellular functional changes similar to those induced by high LDL concentrations. EC incubated with 1-25 uM arachidonic acid had increased H2O2 production and heightened endocytotic activity. Likewise, EC pre-loaded with [3H]arachidonic acid when exposed to increasing LDL levels (90-330 mg/dl cholesterol) had a dose-dependent rise in cytosolic [3H]arachidonic acid. The phospholipase A2 inhibitors, 4-bromophenacyl bromide and 7,7-dimethyleicosadienoic acid, markedly inhibited H2O2 production in EC exposed to 240 mg/dl LDL cholesterol. These findings suggest that arachidonic acid contributes mechanistically to high LDL-perturbed EC H2O2 generation and heightened endocytosis. Such cellular functional changes add to our understanding of endothelial perturbation, which has been hypothesized to be a major contributing factor in the pathogenesis of atherosclerosis.

Atherogenic levels of low-density lipoprotein increase hydrogen peroxide generation in cultured human endothelial cells: possible mechanism of heightened endocytosis.

Cultured human umbilical vein endothelial cells(EC) exposed to atherogenic low-density lipoprotein (LDL) levels have augmented reactive oxygen species generation. Confluent EC were incubated with 30-330 mg/dl LDL cholesterol and cellular hydrogen peroxide (H2O2) generation measured. EC incubated with 30 and 90 mg/dl LDL cholesterol showed similar low level H2O2 production. In contrast, EC exposed to 180 and 330 mg/dl LDL cholesterol have a marked, dose-related elevation in H2O2 generation. Subsequent studies have explored if direct EC exposure to H2O2 promotes cellular functional changes similar to those induced by high LDL levels (> 160 mg/dl cholesterol). Confluent EC were incubated with 0.1-10 mM H2O2 for 30 minutes and endocytosis measured and cytoskeletal structure examined. H2O2 exposure (0.5 and 1 mM) promoted heightened EC endocytosis, which similarly occurs with high LDL exposure. Likewise, cytoskeletal examination of EC perturbed with 1 mM H2O2 reveals structural remodeling with a marked increase in stress fibers, which similarly happens with high LDL levels. The above observations that high LDL levels cause increased EC H2O2 production, and direct H2O2 exposure promotes cellular functional changes similar to those induced by high LDL concentrations, suggest a modulatory role for reactive oxygen species. Thus LDL-induced reactive oxygen species generation may contribute mechanistically to endothelial perturbation, which has been hypothesized to be a major contributing factor in the pathogenesis of atherosclerosis.

Retinoic acid, dibutyryl-cAMP, and differentiation affect the expression of retinoic acid receptors in F9 cells.

Expression of the retinoic acid receptors alpha and beta (RAR-alpha and RAR-beta) was examined in F9 cells, an embryonal carcinoma cell model established for the study of retinoid metabolism and function. Addition of retinoic acid to F9 cell medium caused a dose-dependent increase in RAR-beta mRNA within 3 hr that reached 5- to 30-fold greater than the constitutively expressed mRNA by 24 hr. The elevation in mRNA resulted from increased transcription, as demonstrated by nuclear run-on transcription, did not require protein synthesis, and required the constant presence of retinoic acid. N6,O2'-Dibutyryl-cAMP attenuated the retinoic acid-induced increase in RAR-beta mRNA by a post-transcriptional mechanism. In contrast, RAR-alpha mRNA in F9 stem cells was affected less (1.2- to 1.4-fold increase) by retinoic acid and decreased 3-fold transiently when fresh serum was added to the medium. Differentiation of F9 cells resulted in increased steady-state levels of RAR-beta mRNA in primitive (4-fold), parietal (3-fold), and visceral (8-fold) endoderm but decreased steady-state levels of RAR-alpha mRNA in primitive (2-fold), parietal (3-fold), and visceral (1.4-fold) endoderm. These data demonstrate that RAR-beta is a primary target gene for retinoic acid in a characterized model of retinoid function, indicate that constitutive expression of both RAR-beta and RAR-alpha is dependent upon the differentiation state, and suggest hormonal modulation of RAR-beta by cAMP and modulation of RAR-alpha by serum factors. These results distinguish the effects of serum, cAMP, and retinoic acid on the expression of RAR from the effects mediated by differentiation.

Enhanced expression of c-fos is not obligatory for retinoic acid-induced F9 cell differentiation.

c-fos was studied in F9 cells to determine whether changes in its expression are an early and/or obligatory event in retinoic acid-induced F9 cell differentiation. Induction of c-fos transcripts was not observed at times early or late during retinoic acid-promoted differentiation, but a decrease in c-myc mRNA was noted as early as 1 h after retinoic acid dosing. Induction of a rapid and transient change in c-fos expression in F9 cells was observed only in response to serum stimulation. Therefore, although expression of c-fos may be involved in the cellular growth and proliferation of F9 cells, as indicated by the response to serum, an increase in c-fos is not required for retinoic acid-induced differentiation.