Eisuke Nishio

The involvement of reactive oxygen species and arachidonic acid in α1-adrenoceptor-induced smooth muscle cell proliferation and migration

In a previous study, we demonstrated phenylephrine‐stimulated arachidonic acid (AA) release in rabbit cultured aortic smooth muscle cells. Therefore, we have investigated the functional implications of AA which are involved in the cellular response to phenylephrine, particularly proliferation and migration of rabbit cultured aortic smooth muscle cells. First, to determine whether AA directly modifies proliferation and mobility of vascular smooth muscle cells (VSMCs), we exposed the cells to AA. AA induced proliferation and migration of the cells in a dose‐dependent fashion. Concomitantly added catalase inhibited the proliferation and chemotaxis induced by AA of VSMCs. Conversely, aminotriazole enhanced the proliferation and migration induced by AA. Secondly, we investigated whether the proliferation and migration of VSMCs by phenylephrine were related to AA and hydrogen peroxide (H2O2). The proliferation and chemotaxis of VSMCs by phenylephrine were inhibited by a phospholipase A2 (PLA2) inhibitor, or catalase. Lastly, we investigated the effects of AA and phenylephrine on the content of H2O2 in VSMCs. AA and phenylephrine treatment led to an increase of H2O2 in a dose‐dependent manner. These results suggest that the addition of phenylephrine to the cells caused the enhancement of proliferation and migration, probably by mediating AA release and reactive oxygen species (ROS) production.

Homocysteine as a modulator of platelet-derived growth factor action in vascular smooth muscle cells: a possible role for hydrogen peroxide

1 Homocysteine is an independent risk factor for cardiovascular disease. The mechanisms by which elevated plasma concentrations of homocysteine are related to the pathogenesis of atherosclerosis are not fully understood. Therefore, we examined the effect of homocysteine on cell replication of rat cultured vascular smooth muscle cells (VSMCs) at concentrations similar to those observed in clinical studies. 2 The incorporation of [3H]‐thymidine was used as a marker of mitosis. Homocysteine (250–500 μM) was a weak mitogen as compared to platelet‐derived growth factor‐BB (PDGF‐BB, 1 nM) and serum (10%), but it potentiated the mitogenic effect of PDGF‐BB four fold at 500 μM. This enhancement of mitogenesis was blunted by the addition of the scavenging enzyme catalase or the antioxidant N‐acetyl‐L‐cysteine. 3 Furthermore, stimulation of VSMC with homocysteine (25–500 μM) decreased the glutathione peroxidase activity of the cells to 50% of control at 500 μM. Inversely, homocysteine enhanced the superoxide dismutase (SOD) activity to 137% of control at 500 μM, but it had no effect on the catalase activity. 4 Homocysteine decreased the activity of bovine purified liver cytosolic glutathione peroxidase in a time‐ and dose‐dependent manner. The maximum decrease was 50%. 5 In summary, homocysteine has a weak mitogenic effect on VSMC, but it dramatically enhances the mitogenic response of PDGF‐BB, presumably by disturbing the activity of antioxidant enzymes.

Thrombin activates p38 mitogen-activated protein kinase in vascular smooth muscle cells

Thrombin is a potent mitogen for vascular smooth muscle cells. However, the signaling pathways by which thrombin mediates its mitogenic response are not fully understood. The ERK (extracellular signal-regulated protein kinase) and JNK (c-Jun N-terminal kinase) members of the mitogen-activated protein kinase (MAPK) family are reported to be activated by thrombin. We have investigated the response to thrombin of another member of the MAPK family, p38 MAPK, which has been suggested to be activated by both stress and inflammatory stimuli in vascular smooth muscle cells. We found that thrombin induced time- and dose-dependent activation of p38 MAPK. Maximal stimulation of p38 MAPK was observed after a 10-min incubation with 1 unit ml(-1) thrombin. GF109203X, a protein kinase C inhibitor, and prolonged treatment with phorbol 12-myristate 13-acetate partially inhibited p38 MAPK activation. A tyrosine kinase inhibitor, genistein, also inhibited p38 MAPK activation in a dose-dependent manner. p38 MAPK activation was inhibited by overexpression of betaARK1ct (beta-adrenergic receptor kinase I C-terminal peptide). p38 MAPK activation was also inhibited by expression of dominant-negative Ras, not by dominant-negative Rac. We next examined the effect of a p38 MAPK inhibitor, SB203580, on thrombin-induced proliferation. SB203580 inhibited thrombin-induced DNA synthesis in a dose-dependent manner. These results suggest that thrombin activates p38 MAPK in a manner dependent on Gbetagamma, protein kinase C, a tyrosine kinase, and Ras, that p38 MAPK has a role in thrombin-induced mitogenic response in the cells.

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitor impairs cell differentiation in cultured adipogenic cells (3T3-L1)

Lovastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, inhibits the synthesis of mevalonic acid. We examined the effect of lovastatin on the differentiation of the fibroblast/adipocyte cell line (3T3-L1). Lovastatin inhibits the differentiation of 3T3-L1 cells in a dose-dependent fashion. The inhibitory effect of lovastatin was partially reversed by adding exogenous mevalonic acid to the 3T3-L1 cells. Exogenous cholesterol (15 micrograms/ml) did not prevent lovastatin inhibition of adipocyte conversion. The isoprenoids, farnesol and geraniol, partially prevented lovastatin inhibition of adipocyte conversion but squalene did not prevent lovastatin inhibition of adipocyte conversion. We conclude that the inhibitory effect of lovastatin was partially due to the blockade of the pathway leading to synthesis of isoprenoids, which are downstream products of mevalonic acid.

Nitric oxide donor-induced apoptosis in smooth muscle cells is modulated by protein kinase C and protein kinase A

We have demonstrated previously that exogenously applied nitric oxide (NO) redox species induced apoptosis in smooth muscle cells. The present studies were undertaken to characterize further the potential role of protein kinase C and protein kinase A in the regulation of S-nitroso-N-acetylpenicillamine-induced apoptosis in smooth muscle cells. S-nitroso-N-acetylpenicillamine-induced apoptosis was prevented by the protein kinase C inhibitors, calphostin C and H-7, and was potentiated by protein kinase C activator, phorbol 12-myristate 13-acetate (PMA). Furthermore, S-nitroso-N-acetylpenicillamine prolonged membrane translocation of protein kinase C-epsilon. The membrane permeable analogue, dibutyryl-cAMP (Db-cAMP), potentiated S-nitroso-N-acetylpenicillamine-induced apoptosis, whereas the selective protein kinase A competitive inhibitor, Rp-cAMP, prevented S-nitroso-N-acetylpenicillamine-induced apoptosis. These results indicate that both protein kinase C and protein kinase A are involved in S-nitroso-N-acetylpenicillamine-induced apoptosis in smooth muscle cells.

Aspirin and salicylate enhances the induction of inducible nitric oxide synthase in cultured rat smooth muscle cells

Aspirin and sodium salicylate enhance to a similar extent the production of nitric oxide (NO) in cultured smooth muscle cells following stimulation by interleukin-1beta (IL-1beta). The similar potencies of aspirin and sodium salicylate indicate that acetylation of cellular macromolecules is not essential for the enhancement of NO production. The failure of added prostaglandin E2 (PGE2) or Thromboxane A2 (TXA2) to overcome the effects of aspirin or sodium salicylate indicates that these effects are not simply the result of inhibition of prostaglandin synthesis. The enhancement of NO production occurs dependent of the effects of these agents on induction of inducible nitric oxide synthase (iNOS) expression by IL-1beta. Aspirin and sodium salicylate enhance the induction of iNOS expression by IL-1beta. We previously reported that pretreatment of vascular smooth muscle cells (VSMCs) with high glucose decreased the response of the cells by IL-1beta, that is, the induction of iNOS expression and NO production. We investigated the effect of aspirin and sodium salicylate on the response by IL-1beta of VSMCs pretreated with high glucose (25 mM). Aspirin and sodium salicylate ameliorate the down-regulation of iNOS expression and the decrease of NO production caused by pretreatment with high glucose (25 mM). These results suggest a possible therapeutic role in atherosclerotic disease and diabetes mellitus for aspirin and sodium salicylate by enhancing the level of iNOS expression and NO production.

Cigarette smoke extract is a modulator of mitogenic action in vascular smooth muscle cells

Cigarette smoking is associated with an increased incidence of atherosclerotic disease. In this study, we examined the mechanism underlying the growth-modulating effects of cigarette smoke extract (CSE) in confluent vascular smooth muscle cells (VSMCs). The treatment of VSMC by CSE decreased the activities of superoxide dismutase (SOD), catalase and glutathione peroxidase of VSMC in a time-dependent manner. In mitogenesis assays using the confluent cells, CSE was not a direct mitogen for VSMC, but potentiated the stimulatory effect of hydrogen peroxides. The reduction of activities of catalase and glutathione peroxidase was partially prevented by SH-containing compounds. In summary, CSE enhanced the mitogenic effect response of hydrogen peroxides, largely depending on the dysregulation of the activities of SOD, catalase and glutathione peroxidase by CSE.

Effects of alcohol consumption on antioxidant content and susceptibility of low-density lipoprotein to oxidative modification.

OBJECTIVE The effects of moderate alcohol intake on antioxidant content of low-density lipoprotein (LDL) and the susceptibility of LDL to oxidative modification were examined in 12 healthy adult males. DESIGN Volunteers abstained from alcohol for 3 weeks and then 12 subjects (alcohol intake group) consumed alcohol (0.5 g/kg/day) as brandy for 4 weeks; 4 subjects (control abstinence group) did not consume any alcohol for the entire study. RESULTS In the alcohol intake group, plasma total cholesterol (TC), triglyceride (TG), phospholipids (PL), free cholesterol (FC) and apoprotein B (apoB) levels in LDL decreased significantly after alcohol intake; however, since TC/apoB, TG/apoB, PL/apoB and FC/apoB ratios did not change significantly, it is clear that LDL particle numbers decreased. Vitamin E and vitamin A levels in plasma, and vitamin E content of LDL also did not change significantly. Beta-carotene levels in plasma and in LDL decreased significantly in the alcohol intake group. In the abstinence group, lipid levels and vitamin levels did not change. Lag time before the onset of LDL oxidation and propagation rate of LDL oxidation in the alcohol intake group did not change significantly. CONCLUSIONS Moderate alcohol intake decreases particle numbers of LDL without any changes in chemical composition, vitamin E content and susceptibility of LDL to oxidative modification. However, beta-carotene content was decreased significantly by even moderate alcohol intake.

NO INDUCED APOPTOSIS ACCOMPANYING THE CHANGE OF ONCOPROTEIN EXPRESSION AND THE ACTIVATION OF CPP32 PROTEASE

Previously we have shown that nitric oxide (NO) donors induced apoptosis in vascular smooth muscle cells (VSMCs). However, the mechanisms by which NO induced apoptosis in VSMCs are entirely unknown. In the present study, we intended to identify the mechanism by which NO donors induce apoptosis in VSMCs. First, we evaluated the expression of c-Myc, P53, and Bcl-2 proteins in VSMCs treated by NO donors. c-Myc and P53 protein expression increased after VSMCs were incubated with NO donors for 6 hr and reached a maximum level at 24 hr, while Bcl-2 protein decreased after 12 hr incubation. Next we investigated to see whether the CPP32 protease activation was involved in NO donors-induced apoptosis. In VSMCs treated by NO donors, the increase of CPP32 protease activity was observed and specific inhibition of CPP32 activity significantly prevented apoptosis induced by NO donors in a dose-dependent manner. These results suggest that NO donors induced apoptosis through proto-oncoprotein expression and CPP32-like protease activation.

Role of the lipoxygenase pathway in phenylephrine-induced vascular smooth muscle cell proliferation and migration.

We studied the effects of phenylephrine-stimulated proliferation and migration of vascular smooth muscle cells and the role of 12-lipoxygenase-mediated pathways under normal as well as high glucose conditions. Phenylephrine-induced increases in cellular proliferation and migration were attenuated by the specific 12-lipoxygenase inhibitor baicalein. In contrast, neither of the cyclo-oxygenase inhibitors, indomethacin or ibuprofen, had any effect. Direct addition of the 12-lipoxygenase product, 12-S-hydroxyeicosatetraenoic acid (12-HETE), increased the proliferation and migration of vascular smooth muscle cells treated with both phenylephrine and nordihydroguaiaretic acid. Furthermore, we observed that phenylephrine induced greater increases in the proliferation and migration of vascular smooth muscle cells and also that the 12-lipoxygenase inhibitor prevented the enhancement of proliferation and migration of vascular smooth muscle cells induced by phenylephrine in the presence of high glucose (25 mmol/l). These results suggest that 12-lipoxygenase activation plays a key role in phenylephrine-induced responses of vascular smooth muscle cells under normal and hyperglycemic conditions. 12-lipoxygenase may be a good pharmacological target for treatment of vascular disease of hypertension and diabetes mellitus.