Takatoshi Saito

Inhibition of Rho-Associated Kinase Results in Suppression of Neointimal Formation of Balloon-Injured Arteries

BACKGROUND Rho-associated kinase (ROCK), an effector of small GTPase Rho, regulates vascular tone via a calcium sensitization mechanism and plays a key role in the pathogenesis of hypertension. However, its role in vascular growth remains unclear. METHODS AND RESULTS Y-27632, a specific ROCK inhibitor, and the overexpression of dominant-negative ROCK suppressed the mitogen-induced DNA synthesis of cultured vascular smooth muscle cells (VSMCs), which indicates the essential role of ROCK in the control of VSMC proliferation in vitro. Y-27632 also suppressed the chemotaxis of VSMCs. Male Wistar rats were systemically given Y-27632 (35 to 70 mg. kg(-1). day(-1)) through an intraperitoneal infusion. The neointimal formation of balloon-injured carotid arteries was significantly suppressed in Y-27632-treated rats (intima/media ratio, 0.22+/-0.02) compared with vehicle-treated rats (intima/media ratio, 0.92+/-0.21) or hydralazine-treated rats with a similar blood pressure decrease (intima/media ratio, 1.03+/-0.15). The phosphorylation of myosin phosphatase and myosin light chain was elevated in injured arteries in a Y-27632-sensitive manner, indicating the augmentation of ROCK activity in neointimal formation. The downregulation of the cyclin-dependent kinase inhibitor p27(kip1) in injured vessels was reversed by Y-27632 treatment, reflecting the antiproliferative effect of ROCK inhibition in vivo. CONCLUSIONS We conclude that ROCK plays a key role in the process of neointimal formation after balloon injury. Thus, the inhibition of ROCK may be a potential therapeutic strategy for treating vascular proliferative disorders and hypertension.

Oxidized LDL Regulates Vascular Endothelial Growth Factor Expression in Human Macrophages and Endothelial Cells Through Activation of Peroxisome Proliferator–Activated Receptor-γ

Abstract— Vascular endothelial growth factor (VEGF) has been recognized as an angiogenic factor that induces endothelial proliferation and vascular permeability. Recent studies have also suggested that VEGF can promote macrophage migration, which is critical for atherosclerosis. We have reported that VEGF is remarkably expressed in activated macrophages, endothelial cells, and smooth muscle cells within human coronary atherosclerotic lesions, and we have proposed the significance of VEGF in the progression of atherosclerosis. To clarify the mechanism of VEGF expression in atherosclerotic lesions, we examined the regulation of VEGF expression by oxidized low density lipoprotein (Ox-LDL), which is abundant in atherosclerotic arterial walls. A recent report has revealed that peroxisome proliferator–activated receptor-&ggr; (PPAR&ggr;) is expressed not only in adipocytes but also in monocytes/macrophages and has suggested that PPAR&ggr; may have a role in the differentiation of monocytes/macrophages. Furthermore, 9- and 13-hydroxy-(S)-10,12-octadecadienoic acid (9- and 13-HODE, respectively), the components of Ox-LDL, may be PPAR&ggr; ligands. Therefore, we investigated the involvement of PPAR&ggr; in the regulation of VEGF by Ox-LDL. PPAR&ggr; expression was detected in human monocyte/macrophage cell lines, human acute monocytic leukemia (THP-1) cells, and human coronary artery endothelial cells (HCAECs). Ox-LDL (10 to 50 &mgr;g/mL) upregulated VEGF secretion from THP-1 dose-dependently. VEGF mRNA expression in HCAECs was also upregulated by Ox-LDL. The mRNA expression of VEGF in THP-1 cells and HCAECs was also augmented by PPAR&ggr; activators, troglitazone (TRO), and 15-deoxy-&Dgr;12,14-prostaglandin J2 (PGJ2). In contrast, VEGF expression in another monocyte/macrophage cell line, human histiocytic lymphoma cells (U937), which lacks PPAR&ggr; expression, was not augmented by TRO or PGJ2. We established the U937 cell line, which permanently expresses PPAR&ggr; (U937T). TRO and Ox-LDL augmented VEGF expression in U937T. In addition, VEGF production by THP-1 cells was significantly increased by exposure to 9-HODE and 13-HODE. In conclusion, Ox-LDL upregulates VEGF expression in macrophages and endothelial cells, at least in part, through the activation of PPAR&ggr;.

Down regulation of peroxisome proliferator-activated receptorγ expression by inflammatory cytokines and its reversal by thiazolidinediones

Aims/hypothesis. Previous studies show that inflammatory cytokines play a part in the development of insulin resistance. Thiazolidinediones were developed as insulin-sensitizing drugs and are ligands for the peroxisome proliferator-activated receptorγ (PPARγ). We hypothesized that the anti-diabetic mechanism of thiazolidinediones depends on the quantity of PPARγ in the insulin resistant state in which inflammatory cytokines play a part. Methods. We isolated rat PPARγ1 and γ2 cDNAs and examined effects of various cytokines and thiazolidinediones on PPARγ mRNA expression in rat mature adipocytes. Results. Various inflammatory cytokines, such as tumour necrosis factor-α (TNF-α), interleukin-1α (IL-1α), IL-1β, IL-6 and leukaemia inhibitory factor decreased PPARγ mRNA expression. In addition, hydrogen peroxide, lysophosphatidylcholine or phorbol 12-myristate 13-acetate also decreased the expression of PPARγ. The suppression of PPARγ mRNA expression caused by 10 nmol/l of TNF-α was reversed 60 % and 55 % by treatment with 10–4 mol/l of troglitazone and 10–4 mol/l of pioglitazone, respectively. The suppression of glucose transporter 4 mRNA expression caused by TNF-α was also reversed by thiazolidinediones. Associated with the change of PPARγ mRNA expression, troglitazone improved glucose uptake suppressed by TNF-α. Conclusion/interpretation. Our study suggests that inflammatory cytokines could be factors that regulate PPARγ expression for possible modulation of insulin resistance. In addition, we speculate that the regulation of PPARγ mRNA expression may contribute to the anti-diabetic mechanism of thiazolidinediones. [Diabetologia (1999) 42: 702–710]

Thiazolidinediones, peroxisome proliferator-activated receptor γ agonists, regulate endothelial cell growth and secretion of vasoactive peptides

Insulin resistance has been highlighted as a common causal factor for glucose intolerance, hypertension and dyslipidemia, all of which are cardiovascular risk factors. A new class of antidiabetic agents, thiazolidinediones (TZDs), has been developed and demonstrated to improve insulin sensitivity. TZDs are high affinity ligands for peroxisome proliferator-activated receptor gamma (PPARgamma), the crucial transcription factor for adipocytes. Recent studies showed that PPARgamma is also expressed in monocytes/macrophages and is suggested to be involved in atherosclerosis. We could detect PPARgamma gene transcript in several cultured endothelial cells (human aortic endothelial cells (HAoECs), human coronary artery endothelial cells (HCAECs), human umbilical vein endothelial cells (HUVECs) and bovine carotid artery endothelial cells (BAECs)) as well as human coronary arteries we examined. Since endothelial dysfunction is critical for atherosclerosis, we investigated the effects of TZDs, troglitazone (TRO) and pioglitazone (PIO), on endothelial cell growth and secretion of C-type natriuretic peptide (CNP), which we demonstrated as a novel endothelium-derived relaxing peptide, and endothelin (ET), a potent vasoconstrictor, using HAoECs, HCAECs, HUVECs and BAECs. When all these cultured endothelial cells were daily treated with TRO and PIO for 5 days, both TRO and PIO (10(-8)M) significantly stimulated (3)H-thymidine incorporation of all these endothelial cells. In contrast, higher dose of TRO and PIO (10(-5)M) significantly suppressed DNA synthesis. TRO and PIO also exerted the compatible effect on the increase of cell numbers. TRO and PIO significantly enhanced CNP secretion from BAECs. In contrast, ET secretion from BAECs was suppressed by both TRO and PIO in a dose-dependent manner. The results of the present study suggest that TZDs modulate endothelial functions, including regulation of endothelial cell growth and secretion of endothelium-derived vasoactive substances, which affect vascular tone and remodeling in the process of atherosclerosis.

Oxidative stress augments secretion of endothelium-derived relaxing peptides, C-type natriuretic peptide and adrenomedullin

Objective Excess oxidative stress is one of the major metabolic abnormalities on vascular walls in hypertension and atherosclerosis. In order to further elucidate the endothelial function under oxidative stress, the effect of hydrogen peroxide (H2O2) on expression of two novel endothelium-derived vasorelaxing peptides, C-type natriuretic peptide (CNP) and adrenomedullin (AM) from bovine carotid artery endothelial cells (BCAECs) was examined. Methods BCAECs were treated with H2O2 (0.1-1.0 mmol/l) and/or an antioxidant, N-acetylcysteine (NAC) (5–10 mmol/l), and incubated for 48 h. The concentrations of CNP and AM were measured with the specific radioimmun assays that we originally developed. CNP and AM mRNA expressions were also examined by reverse transcription-polymerase chain reaction (RT-PCR). Results Treatment of BCAECs with 0.5 and 1 mmol/l H2O2 induced 9-and 10-fold increases of CNP concentration in the media. Addition of 10 mmol/l NAC significantly suppressed the effect of H2O2 by 52%. RT-PCR analysis showed that CNP mRNA expression in BCAECs was also rapidly augmented within 1 h with H2O2 (1 mmol/l) treatment, and reached a peak at 3 h to show a 10-fold increase. AM secretion from BCAECs also increased to two-fold with exposure to 0.5 mmol/l H2O2, accompanied with the augmented level of AM mRNA. NAC 10 mmol/l completely suppressed the effect of H2O2 on AM secretion. Conclusions In this study, it has been demonstrated that H2O2 augments endothelial secretion of the two endothelium-derived relaxing peptides, CNP and AM. Our findings suggest the increased secretion of CNP and AM from endothelium under oxidative stress may function to compensate the impaired nitric oxide-dependent vasorelaxation in hypertension and atherosclerosis.

C-Type Natriuretic Peptide Induces Redifferentiation of Vascular Smooth Muscle Cells With Accelerated Reendothelialization

Abstract—We recently reported that C-type natriuretic peptide (CNP) occurs in vascular endothelial cells and acts as a vascular-type natriuretic peptide. In the present study, we stimulated the cGMP cascade in proliferating smooth muscle cells (SMCs), in which particulate guanylate cyclase-B, the specific receptor for CNP, is predominantly expressed, by use of an adenovirus encoding rat CNP cDNA (Ad.CNP). In the Ad.CNP-treated cultured SMCs, CNP caused the growth inhibition of SMCs at G1 phase with an early increase of p21CIP1/WAF1 expression and subsequent upregulation of p16INK4a. The expression of smooth muscle myosin heavy chain-2, which is the molecular marker of highly differentiated SMCs, was reinduced in the Ad.CNP- treated SMCs. The Ad.CNP-treated SMCs also reexpressed particulate guanylate cyclase-A, which shows high affinity to atrial and brain natriuretic peptide and is exclusively expressed in well-differentiated SMCs. CNP, which was overexpressed in rabbit femoral arteries in vivo at the time of balloon injury, significantly suppressed neointimal formation. Furthermore, an enhancement of the expression of smooth muscle myosin heavy chain-2 occurred in the residual neointima. In addition, early regeneration of endothelial cells was observed in the Ad.CNP-infected group. Thus, stimulation of cGMP cascade in proliferating dedifferentiated SMCs can induce growth inhibition and redifferentiation of SMCs with accelerated reendothelialization.

Shear stress attenuates endothelin and endothelin-converting enzyme expression through oxidative stress.

Shear stress is known to dilate blood vessels and exert an antiproliferative effect on vascular walls. These effects have partly been ascribed to shear stress-induced regulation of the secretion of endothelium-derived vasoactive substances. In this study, to elucidate the role of shear stress in endothelin production by endothelial cells, we examined the effect of physiological shear stress on the mRNA expression of endothelin-converting enzyme-1 (ECE-1) as well as endothelin-1 (ET-1) in cultured bovine carotid artery endothelial cells (BAECs) and human umbilical vein endothelial cells (HUVECs), using a parallel plate-type flow chamber. ECE-1 mRNA expression was significantly down-regulated by shear stress in an intensity- and time-dependent manner within the physiological range (1.5 to 15 dyn/cm(2)). ET-1 mRNA expression decreased together with ECE-1 mRNA expression. Shear stress at 15 dyn/cm(2) for 30 min induced a significant increase in the intracellular peroxide concentration, and the down-regulation of ECE-1 and ET-1 mRNA expression by shear stress was attenuated almost completely on treatment with N-acetyl cysteine (NAC), an antioxidant (20 mM). Furthermore, when H(2)O(2) (0.5 to 2 mM) was added to BAECs in static culture, the ECE-1 as well as ET-1 mRNA expression was attenuated in proportion to the concentration of H(2)O(2). It is suggested that endothelial cells sense shear stress as oxidative stress and transduce signal for the regulation of the gene expression of ECE as well as ET to attenuate vascular tone and inhibit the proliferation of vascular smooth muscle cells.

Physiologic shear stress suppresses endothelin-converting enzyme-1 expression in vascular endothelial cells

Shear stress dilates blood vessels and exerts an antiproliferative effect on vascular walls. These effects are ascribed to shear stress-induced, endothelium-derived vasoactive substances. Endothelin-converting enzymes (ECEs), the enzymes that convert big endothelin-1 (ET-1) to ET-1, have recently been isolated and the corresponding proteins have been termed ECE-1 and ECE-2. Furthermore, two isoforms of human ECE-1 have been demonstrated and termed ECE-1 alpha and ECE-1 beta. In this study, to elucidate the role of ECE-1 under shear stress we examined the effect of physiologic shear stress on the mRNA expression of ECE-1 and ET-1 in cultured bovine carotid artery endothelial cells (BAECs) and human umbilical veins (HUVECs), and also ECE-1 alpha mRNA expression in HUVECs. ECE-1 mRNA expression was significantly downregulated by shear stress in 24 h, both in BAECs and HUVECs, in a shear stress intensity-dependent manner. The expression of ECE-1 alpha mRNA was also attenuated by shear stress in HUVECs. ET-1 mRNA expression showed a concordant decrease with ECE-1 mRNA expression. These results suggest that shear stress-induced gene regulation of ET-1 and ECE-1 mRNA expression can contribute to the decrease of ET-1 peptide level by shear stress.

Oxidative stress suppresses the endothelial secretion of endothelin

To address endothelial function on vascular walls exposed to oxidative stress, we investigated the effect of oxidative stress on the secretion of endothelin-1 (ET-1) from cultured bovine carotid artery endothelial cells (BAECs). Concentrations of ET-1 in the media were measured by a specific radioimmunoassay and ET-1 mRNA expression was estimated by Northern blot analysis. Treatment of BAECs with 0.5-2.0 mM H2O2 for 3 h suppressed both ET-1 secretion and ET-1 mRNA expression in a dose-dependent manner compared to control. Attenuation of ET-1 mRNA expression by H2O2 was revealed to take place at the transcriptional level. The addition of NG-nitro-L-arginine-methyl ester (L-NAME) 10 microns, a specific nitric oxide synthase inhibitor, had no effect on H2O2-induced suppression of ET-1 mRNA expression. Suppression of ET secretion under oxidative stress observed in the present study is proposed to be a compensatory mechanism of endothelial cells to inhibit vasoconstriction and proliferation during oxidative stress.

Angiotensin II suppresses growth arrest specific homeobox (Gax) expression via redox-sensitive mitogen-activated protein kinase (MAPK).

Oxidative stress is known to be involved in growth control of vascular smooth muscle cells (VSMCs). We and others have demonstrated that angiotensin II (Ang II) has an important role in vascular remodeling. Several reports suggested that VSMC growth induced by Ang II was elicited by oxidative stress. Gax, growth arrest-specific homeobox is a homeobox gene expressed in the cardiovascular system. Over expression of Gax is demonstrated to inhibit VSMC growth. We previously reported that Ang II down-regulated Gax expression. To address the regulatory mechanism of Gax, we investigated the significance of oxidative stress in Ang II-induced suppression of Gax expression. We further examined the involvement of mitogen-activated protein kinases (MAPKs), which is crucial for cell growth and has shown to be activated by oxidative stress, on the regulation of Gax expression by Ang II. Ang II markedly augmented intracellular H2O2 production which was decreased by pretreatment with N-acetylcystein (NAC), an anti-oxidant. Ang II and H2O2 decreased Gax expression dose-dependently and these effects were blocked by administration of both NAC and pyrrolidine dithiocarbamate (PDTC), another anti-oxidant. Ang II and H2O2 induced marked activation of extracellular signal-responsive kinase1/2 (ERK1/2), which was blocked by NAC. Ang II and H2O2 also activated p38MAPK, and they were blocked by pre-treatment with NAC. However, the level of activated p38MAPK was quite low in comparison with ERK1/2. Ang II- or H2O2 -induced Gax down-regulation was significantly inhibited by PD98059, an ERK1/2 inhibitor but not SB203580, a p38MAPK inhibitor. The present results demonstrated the significance of regulation of Gax expression by redox-sensitive ERK1/2 activation.