Michiyo Ikeda

Interleukin 6 gene transcripts are expressed in human atherosclerotic lesions

Factors controlling the proliferation of vascular smooth muscle cells (SMC) are thought to be key elements in the progression of atherosclerosis. We have previously shown that interleukin 6 (IL-6) stimulates the growth of SMC in vitro and that IL-6 gene transcripts are expressed in atherosclerotic lesions of genetically hyperlipidemic rabbits. To understand the involvement of IL-6 in the development of human atherosclerosis, we investigated IL-6 mRNA expression in atherosclerotic arteries from patients undergoing surgical vascularization, utilizing reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization analyses. In RT-PCR analysis, the atherosclerotic arteries showed 10- to 40-fold levels of IL-6 mRNA expression over the non-atherosclerotic artery. In in situ hybridization analysis, IL-6 gene transcripts were observed in the thickened intimal layer of atherosclerotic lesions. These results strongly suggest the involvement of IL-6 in the development of human atherosclerosis.

Homocysteine Increases Nitric Oxide Synthesis in Cytokine-Stimulated Vascular Smooth Muscle Cells

BACKGROUND Elevated plasma homocysteine levels have been reported to be an independent risk factor for vascular disease. However, there have been no reports concerning the effects of homocysteine on the production of nitric oxide (NO), another modulator of vascular function and proliferation, by the vascular smooth muscle. METHODS AND RESULTS We investigated the effects of homocysteine on NO synthesis by measuring the production of nitrite, a stable metabolite of NO, in cultured rat vascular smooth muscle cells (VSMCs). Incubation of cultures with interleukin (IL)-1beta 10 ng/mL for 24 hours caused a significant increase in nitrite generation. The IL-1beta-induced nitrite production by VSMCs was significantly increased by homocysteine in a dose-dependent manner. This effect of homocysteine was significantly inhibited in the presence of NG-monomethyl-L-arginine or actinomycin D. The homocysteine-induced nitrite production was accompanied by increased inducible NO synthase mRNA and protein accumulation. Cysteine, glutathione, or hydrogen peroxide also increased nitrite accumulation in IL-1beta-stimulated VSMCs. Coincubation with the radical scavenger catalase or superoxide dismutase markedly reduced homocysteine-induced nitrite accumulation. CONCLUSIONS Homocysteine enhances NO synthesis in IL-1beta-stimulated VSMCs, and oxidative products are involved in the effect of homocysteine.

Lipophilic statins augment inducible nitric oxide synthase expression in cytokine-stimulated cardiac myocytes.

&NA; Nitric oxide production by inducible nitric oxide synthase (iNOS) may play an important role in the pathogenesis of cardiovascular dysfunction. We investigated the effects of statins on iNOS expression and subsequent nitric oxide synthesis in cardiac myocytes and the mechanism by which statins exert their effects. We measured the production of nitrite, a stable metabolite of nitric oxide, in cultured neonatal rat cardiac myocytes with the Griess reagent. iNOS mRNA and protein expression were assayed by reverse transcription polymerase chain reaction and Western blotting, respectively. The lipophilic statins fluvastatin and lovastatin significantly increased interleukin‐1&bgr;‐induced nitrite production by cardiac myocytes, whereas hydrophilic pravastatin did not. Increased nitrite production by fluvastatin was accompanied by increased iNOS mRNA and protein accumulation. Exogenous mevalonate, but not squalene, significantly blocked the stimulatory effect of fluvastatin on nitrite production. Cotreatment with geranylgeranylpyr‐ophosphate also reversed the effect of fluvastatin. Furthermore, both Rho inhibitor C3 exoenzyme and Rho kinase inhibitor Y‐27632 significantly increased interleukin‐1&bgr;‐induced nitrite accumulation in cardiac myocytes. These results demonstrated that lipophilic statins upregulate iNOS expression and subsequent nitric oxide formation in cardiac myocytes via inhibition of Rho.

Fluvastatin Upregulates Inducible Nitric Oxide Synthase Expression in Cytokine-Stimulated Vascular Smooth Muscle Cells

Nitric oxide (NO) production by inducible NO synthase (iNOS) may play an important role in the pathogenesis of atherosclerosis. Although fluvastatin has been shown to reduce progression of atherosclerosis, it is not known whether it regulates iNOS expression. We investigated the effects of fluvastatin on iNOS expression and subsequent NO synthesis in vascular smooth muscle cells (VSMCs) and the mechanism by which fluvastatin exerts its effects. Fluvastatin significantly increased interleukin-1&bgr; (IL-1&bgr;)–induced nitrite production by VSMCs in a time-dependent (0 to 24 hours) and dose-dependent (10−8 to 10−5 mol/L) manner. Increased nitrite production by fluvastatin was accompanied by increased iNOS mRNA and protein accumulation. IL-1&bgr; induced nuclear factor-&kgr;B activation in VSMCs, which was not affected by fluvastatin. Exogenous mevalonate significantly prevented the stimulatory effect of fluvastatin on nitrite production. Cotreatment with geranylgeranyl-pyrophosphate also reversed the effect of fluvastatin. Furthermore, both Rho inhibitor C3 exoenzyme and Rho kinase inhibitor Y-27632 significantly increased IL-1&bgr;–induced nitrite accumulation in VSMCs. These results demonstrated that fluvastatin upregulates iNOS expression and subsequent NO formation in rat VSMCs through inhibition of Rho.

Neutrophil Adherence to Rat Cardiac Myocyte by Proinflammatory Cytokines

Summary: Cytokine induction of intercellular adhesion molecule-1 (ICAM-1) in cardiac myocytes may be a critical step in inflammation associated with ischemia–reperfusion injury. We investigated the involvement of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), and interleukin 8 (IL-8) on neutrophil-myocyte adhesion; These cytokines are increased in plasma of patients with acute myocardial infarction (AMI). ICAM-1 expression on cultured neonatal rat cardiac myocytes was determined through immunohistochemical and enzyme-linked immunosorbent assay (ELISA) analysis. ICAM-1 mRNA expression in myocytes was investigated by Northern blot hybridization. Rat neutrophils isolated from peripheral blood (PB) were used for adherence assay. In immunohistochemical study, cultured neonatal rat cardiac myocytes constitutively expressed ICAM-1 molecules. In ELISA analysis, ICAM-1 molecule expression on myocytes was significantly stimulated by TNF-α (100 U/ml), but not by IL-6 (100 U/ml) or IL-8 (100 ng/ml) dose dependently. The effect of TNF-α was observed as early as 6 h after stimulation. Levels of ICAM-1 mRNA were very low or almost undetectable in unstimulated myocytes, but its expression was markedly induced after exposure to TNF-α for 3 h. IL-6 and IL-8 showed no effect on ICAM-1 mRNA accumulation. Adhesion of rat neutrophils to myocytes was stimulated by TNF-α, and the effect of TNF-α on adherence was significantly inhibited by an anti-ICAM-1 monoclonal antibody (MoAb). These results show that TNF-α, but not IL-6 and IL-8, promotes neutrophil-myocyte adhesion through ICAM-1 expression, suggesting involvement of TNF-α in inflammation associated with ischemia–reperfusion injury.

Expression of intercellular adhesion molecule-1 on rat vascular smooth muscle cells by pro-inflammatory cytokines

Infiltration of mononuclear cells is an early pathological finding in human and experimental atherosclerosis. However, the cellular and molecular basis for cell infiltration is incompletely understood. While the intercellular adhesion molecule-1 (ICAM-1) is expressed on endothelial cells and promotes the adhesion of mononuclear cells, there is little information on the expression of ICAM-1 on vascular smooth muscle cells (SMC). In this study, we investigated the expression of ICAM-1 on cultured rat SMC and its regulation by pro-inflammatory cytokines, interleukin 1 alpha (IL-1 alpha), interleukin 6 (IL-6) and monocyte chemoattractant protein 1 (MCP-1). In immunohistochemical staining, ICAM-1 molecules were constitutively expressed on the surface of SMC. In flow cytometric and ELISA analyses, ICAM-1 molecule expression on SMC was significantly upregulated by IL-1 alpha and MCP-1, but not by IL-6, in a dose-dependent manner. The effects of IL-1 alpha and MCP-1 were observed as early as 4 h. In Northern blot analysis, ICAM-1 mRNA was slightly detectable in unstimulated SMC, but its expression was clearly observed following exposure to IL-1 alpha or MCP-1. These results suggest that ICAM-1 on SMC, as well as on endothelial cells, could participate in the focal accumulation of mononuclear cells in human atherosclerotic lesions.

Mitogenic action of interleukin-1α on vascular smooth muscle cells mediated by PDGF

Abstract We have investigated the effect of interleukin-1 (IL-1) on the growth of vascular smooth muscle cells (VSMC) isolated from rat aortae. Murine recombinant IL-la increased tritiated leucine incorporation into VSMC. IL-1 also stimulated tritiated thymidine uptake by VSMC in a dose-dependent manner. On the other hand, Ca 2+ -channel blocker, verapamil, inhibited the IL-1-induced thymidine uptake by VSMC with an IC 50 of 10 −8 M. Antibody specific for platelet-derived growth factor (PDGF) also totally inhibited the IL-1-induced thymidine uptake. IL-1 showed no effects on the intracellular CaZ+ level in VSMC. Above results support the premise that IL-1 promotes the growth of VSMC via induction of endogenous PDGF production and might thus participate in the abnormal proliferation of VSMC that occurs early in atherogenesis.

Involvement of LDL receptor in the proliferation of vascular smooth muscle cells

To study the involvement of the low-density lipoprotein (LDL) receptor in the growth of vascular smooth muscle cells (VSMC), we compared the proliferation of cultured VSMC from Watanabe heritable hyperlipidemic (WHHL) rabbits, which lack the LDL receptor, and VSMC from normal Japanese white rabbits in response to platelet derived growth factor (PDGF). The increase in the number of VSMC from WHHL rabbits in response to PDGF (10(-8) M) was significantly lower than that of VSMC from normal rabbits. PDGF stimulated the synthesis of DNA in VSMC from both normal rabbits and WHHL rabbits, but the response was significantly lower in the latter. To determine the involvement of the LDL receptor in the decreased mitogenic response of WHHL rabbit VSMC, we used an anti-LDL receptor monoclonal antibody (MAb) to normal rabbit VSMC; DNA synthesis of VSMC was stimulated by PDGF, but the effect was significantly blocked by the anti-LDL receptor MAb. Mitogen-activated protein (MAP) kinase activity in normal rabbit VSMC was increased by exposure to PDGF, but the effect was significantly suppressed in the presence of the MAb. The anti-LDL receptor MAb markedly inhibited LDL binding to the surface of normal rabbit VSMC. These results suggest that the LDL receptor influences the proliferation of VSMC and thus might be involved in the pathogenesis of atherosclerosis.

Monocyte–vascular smooth muscle cell interaction enhances nitric oxide production

OBJECTIVE The adhesive interaction of monocytes and vascular smooth muscle cells (VSMCs) has been suggested to be a regulatory signal in the cellular activation that is involved in the pathogenesis of atherosclerosis. We investigated the effects of monocyte-VSMC interaction on inducible nitric oxide (NO) synthase expression. METHODS NO production by the cultured cells was determined by measuring the nitrite content of the culture media using the Griess reagent. The expression of inducible NO synthase protein was assayed by Western blotting. RESULTS Interleukin-1 beta (IL-1 beta) induced nitrite production by VSMCs in a time-dependent manner. The addition of the mouse monocyte cell line J774 to IL-1 beta-stimulated VSMCs further increased nitrite production in a monocyte number-dependent manner. Enhanced nitrite production by coculture was accompanied by increased inducible NO synthase protein accumulation. Addition of tumor necrosis factor-alpha (TNF-alpha) also enhanced IL-1 beta-induced nitrite production by VSMCs, but TNF-alpha showed no effect in the presence of monocytes. Coculture of monocytes and VSMCs in the presence of IL-1 beta secreted substantial amounts of TNF-alpha. The production of nitrite by coculture was markedly inhibited by an anti-TNF-alpha antibody. CONCLUSIONS The present study revealed that direct cell-to-cell interaction between monocytes and VSMCs enhances NO production, suggesting an important role for their interaction in the pathogenesis of atherosclerosis.

Regulation of MHC Class I Expression by Inflammatory Cytokines in Rat Mesangial Cells

We investigated the regulation of major histocompatibility complex (MHC) class I expression by inflammatory cytokines and nitric oxide (NO) in rat mesangial cells by enzyme-linked immunosorbent assay and flow cytometry. MHC class I molecule expression on mesangial cells was significantly stimulated by interferon gamma, tumor necrosis factor alpha and interleukin 1beta, but not by interleukin 6, in a dose-dependent manner. Addition of SIN-1, an NO donor, did not affect the expression of cytokine-induced MHC class I expression. These results suggest that under inflammatory conditions mesangial cells may act as antigen-presenting cells in response to stimulation by cytokines and may be involved in the pathogenesis of immune-mediated glomerular disease.