Abstract-Nltnc oxide (NO) plays an important role not only m the regulation of blood vessel tone, but also m the growth of vascular smooth muscle cells (VSMC) The precise mechamsm involved m the mhlbltlon of VSMC growth by NO 1s not known To further explore the effect of NO on VSMC growth, we examined the effect of NO on the expression of anglotensm II type 1 receptor (AT,-R) that Key Words: vascular smooth muscle cells n anglotensm II receptor w NO w gene transcnptlon P rohferatlon of vascular smooth muscle cells (VSMC) contnbutes to pathological changes of vascular wall such as vascular remodeling, medial hyperplasla, and neomtlmal formation associated with hypertension, atherosclerosis, and vascular qury ' In addition, VSMC produce matrix component of vascular wall such as collagen, fibronectm, and growth factors such as platelet-denved growth factor (PDGF) The matnx and growth factors also play important roles m the process of vascular remodehng and atherogenesls Therefore, understanding the mechanism of VSMC prohferatlon 1s of great mterest m vascular biology and chmcal mvestlgatlon Nltnc oxide (NO) 1s produced by a vanety of mammahan cells such as endothehum, neuron, macrophages, and VSMC fi-om L-arguune by NO synthase (NOS) *J Release of NO stimulates soluble guanylyl cyclase, lea&ng to an increase of intracellular cychc guanosine monophosphate (cGMP) level NO dilates blood vessel and inhibits prohferatlon of VSMC and platelet aggregation 3 These propemes are anti-atherogemc, and decreased NOS actnq 1s beheved to be one of the important feature of early atherogemc process ' Supplementahon of L-argmme, the precursor of NO, lessens the extent of atherosclerosis in diet-induced hypercholesterolermc rabbit 4 In vlvo transfer of type III NOS gene Into the balloon-injured artery decreased neolntlmal VSMC prohferahon 5 However, the precise mechamsm of antl-atherogemc property of NO 1s not completely understood Anglotensm (Ang) II 1s an important vasoactlve peptlde and regulates blood pressure, fluld homeostasls, and electrolyte balance by vasoconstnctlon, faclhtatlon of adrenergc nerve actlvlty, and secretion of aldosterone from the adrenal gland " Recent studies have shown that Ang II IS a growth factor of VSMC and endothehum and plays an important role m atherosclerosis The physlologcal function of Ang II IS transmitted mto target cells via its specific receptor located m the cell membrane There are two lsoforrns for Ang II receptor, which are designated as type 1 receptor (AT,-R)" and type 2 receptor (AT,-R) '" " Losartan that binds to AT,-R and PD123319 that binds to AT*-R are considered to be lsoform speclfk antagonist Although emergmg evidences suggest that AT,-R also plays an important role m the regulation of blood pressure,12 growth mhlbltlon," and apoptosa,'4 most of the cardiovascular effects of Ang II has been believed to be medlated by AT,-R"In rodents, two subtypes of AT, receptor are cloned and named AT,a and AT,b I5 Cultured VSMC express only AT,a-R, and Ang II induces PDGF-A chain, transform...

Abstract-Recent studies suggest that atherosclerosis is a kind of inflammatory process and that cytokine plays important roles in this process. Although it is generally accepted that angiotensin II (Ang II) plays an important role in atherogenesis, the role of Ang II in cytokine production has not been explored. In this report, we investigated the effect of Ang II on the production of interleukin-6 (IL-6), which is a multifunctional proinflammatory cytokine in rat vascular smooth muscle cells. Ang II significantly increased the expression of IL-6 mRNA and protein in a dose-dependent manner (10 Ϫ10 to 10 Ϫ6 mol/L). The expression of IL-6 mRNA induced by Ang II showed 2 peaks at 30 minutes and 12 to 24 hours after stimulation. The effect of Ang II on IL-6 release and mRNA expression was completely blocked by an Ang II type 1 receptor antagonist, CV11974; however, an Ang II type 2 receptor antagonist, PD123319, showed no effect. Chelating of intracellular Ca 2ϩ with BAPTA-AM, inhibition of tyrosine kinase with genistein, and inhibition of mitogen-activated protein kinase kinase with PD98059 completely abolished the effect of Ang II. However, downregulation of protein kinase C by pretreatment with a phorbol ester for 24 hours or a specific protein kinase C inhibitor, calphostin C, did not affect the Ang II-induced expression of IL-6 mRNA. Deletion and mutational analysis of IL-6 gene promoter showed that cAMP-responsive element was important for Ang II-induced IL-6 gene expression. Gel mobility shift assay showed an increase of cAMP-responsive element binding protein by Ang II. These results provide new insights into Ang II signaling and the role of Ang II in the progression of inflammatory changes of blood vessels. (Hypertension. 1999;34:118-125.)

Abstract-Recently, it was shown that Rho-kinase plays an important role in blood pressure regulation. However, it is not known whether Rho-kinase is involved in atherogenesis. Monocyte chemoattractant protein-1 (MCP-1) is an important chemokine that regulates monocyte recruitment and atherogenesis. Therefore, we examined the role of Rho and Rho-kinase in the angiotensin (Ang) II-induced expression of MCP-1. Ang II dose-and time-dependently enhanced the expression of MCP-1 mRNA and the protein production in vascular smooth muscle cells. CV11974, an Ang II type 1 receptor (AT 1 -R) specific antagonist inhibited the enhancement of MCP-1 expression by Ang II, suggesting that the effect of Ang II is mediated by the AT 1 -R. Botulinum C3 exotoxin, a specific inhibitor of Rho, suppressed Ang II-induced MCP-1 production. Key Words: angiotensin II Ⅲ peptides Ⅲ muscle, smooth, vascular Ⅲ receptors, angiotensin Ⅲ kinase A ngiotensin (Ang) II has been known to regulate blood pressure, fluid homeostasis, and electrolyte balance. 1 Recent studies have shown that Ang II plays an important role in atherogenesis as well. The physiological functions of Ang II are transmitted into target cells through its specific receptor located in the plasma membrane. Although there are 2 isoforms for the Ang II receptor, the Ang II type 1 receptor (AT 1 -R) 2 and the Ang II type 2 receptor (AT 2 -R), 3 most of the cardiovascular effects of Ang II are ascribed to AT 1 -R. Vascular smooth muscle cells (VSMCs) express AT 1 -R, and Ang II induces the production of growth factors and extracellular matrices through this receptor. We have recently reported that Ang II induced interleukin-6 production in VSMCs and have proposed that the Ang II-induced cytokine plays an important role in the progression of atherosclerosis. 4 Invasion of monocytes into the blood vessel wall is one of the early steps in the development of atherosclerosis. Various cytokines, such as monocyte chemoattractant protein-1 (MCP-1), 5 macrophage inflammatory protein-1, 6 and RANTES (regulated on activation, normal T-expressed and -secreted), 7 are known to regulate the movement of monocytes. Among these factors, MCP-1 is one of the most potent chemoattractants for monocytes or macrophages in vitro and in vivo. MCP-1 expression is induced in response to tumor necrosis factor-␣ or ␥-interferon, 8 thrombin, 9 or interleukin-1␤ 10 in VSMCs. Pathological conditions such as hypercholesterolemia and vascular injury also induce expression of the MCP-1 gene in the vascular wall. Recent studies have suggested that MCP-1 is critical for the progression of atherosclerosis. Targeted disruption of the receptor for MCP-1 (CCR2) attenuated the development of atherosclerosis when the mice were crossed with apoE knockout mice that develop severe atherosclerosis. 11 Rho-kinase, identified as a downstream target of Rho A, has been shown to phosphorylate the myosin-binding subunit of myosin light chain phosphatase and enhance smooth muscle contraction. 12,13 Y-27632, a specific inhibitor of Rho-kina...

Background-Peroxisome proliferator-activated receptor ␥ (PPAR␥) activators, such as troglitazone (Tro), not only improve insulin resistance but also suppress the neointimal formation after balloon injury. However, the precise mechanisms have not been determined. Angiotensin II (Ang II) plays crucial roles in the pathogenesis of atherosclerosis, hypertension, and neointimal formation after angioplasty. We examined the effect of PPAR␥ activators on the expression of Ang II type 1 receptor (AT 1 -R) in cultured vascular smooth muscle cells (VSMCs). Methods and Results-AT 1 -R mRNA and AT 1 -R protein levels were determined by Northern blot analysis and radioligand binding assay, respectively. Natural PPAR␥ ligand 15-deoxy-⌬ 12,14 -prostaglandin J 2 , as well as Tro, reduced the AT 1 -R mRNA expression and the AT 1 -R protein level. The PPAR␥ activators also reduced the calcium response of VSMCs to Ang II. PPAR␥ activators suppressed the AT 1 -R promoter activity measured by luciferase assay but did not affect the AT 1 -R mRNA stability, suggesting that the suppression occurs at the transcriptional level. Conclusions-PPAR␥ activators reduced the AT 1 -R expression and calcium response to Ang II in VSMCs. Downregulation of AT 1 -R may contribute to the inhibition of neointimal formation by PPAR␥ activators. (Circulation.

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Abstract-Thrombin is a potent mitogen for vascular smooth muscle cells (VSMCs) and plays an important role in the progression of atherosclerosis. Although recent reports have suggested that cAMP response element-binding protein (CREB) is necessary for the survival of neuronal cells, the role of CREB in VSMC proliferation is not determined. We examined the role of CREB in thrombin-induced VSMC proliferation and the effect of thrombin on phosphorylation of CREB at Ser133, which is a critical marker for activation by Western blot analysis. Thrombin induced phosphorylation of CREB in a dose-dependent manner. An oligopeptide, SFLLRN, which activates the thrombin receptor, also induced the phosphorylation of CREB. Inhibition of extracellular signal-regulated protein kinase or inhibition of p38 mitogen-activated protein kinase suppressed the thrombin-induced CREB phosphorylation. Inhibition of the epidermal growth factor receptor by AG1478 also inhibited the thrombin-induced CREB phosphorylation. Overexpression of the dominant-negative form of CREB inhibited thrombin-induced c-fos mRNA expression and incorporation of

Abstract-The plasma level of interleukin-6 (IL-6) is elevated in patients with acute coronary syndromes and has prognostic value. Thrombin is a potent mitogen for vascular smooth muscle cells (VSMCs) and plays an important role in the progression of atherosclerosis. We examined the mechanism of thrombin-induced IL-6 expression in VSMCs.Thrombin induced IL-6 mRNA and protein expression in a dose-dependent manner. Pharmacological inhibition of extracellular signal-regulated protein kinase (ERK), p38 mitogen-activated protein kinase (MAPK), or epidermal growth factor receptor (EGF-R) suppressed the thrombin-induced IL-6 expression. Deletion and mutation analysis of the promoter region of the IL-6 gene by using luciferase as a reporter showed that the DNA segment between Ϫ228 and Ϫ150 bp containing the cAMP response element (CRE) site played a critical role. Thrombin also induced phosphorylation of CRE binding protein (CREB) in an ERK-and a p38 MAPK-dependent manner. Overexpression of the dominant-negative form of CREB inhibited thrombin-induced IL-6 mRNA expression. These results suggest that the CRE site and CREB play an important role in thrombin-induced IL-6 gene expression in VSMCs. Transactivation of EGF-R and activation of ERK and p38 MAPK are involved in this process. CREB may be a novel transcription factor that regulates thrombin-induced gene expression.

Abstract-All-trans retinoic acid (atRA) is a biologically active metabolite of vitamin A that plays an important role in cell differentiation and proliferation. Although neointimal formation after balloon injury of rat carotid artery is inhibited by atRA, the mechanisms are not clearly understood. Because the renin-angiotensin system is one of the crucial components of atherosclerosis, we examined the effects of atRA on the expression of angiotensin II type 1 receptor (AT 1 -R) in vascular smooth muscle cells. atRA (1 mol/L) decreased the AT 1 -R mRNA level by 50% after 24 hours; AT 1 -R number was also reduced to the same extent after 48 hours. atRA markedly suppressed promoter activity of the AT 1 -R promoter-luciferase construct, but AT 1 -R mRNA stability was not affected. Cycloheximide blocked the atRA-induced decrease in AT 1 -R mRNA expression, suggesting that this process requires de novo protein synthesis. Simultaneous treatment with an agonist (Ro40-6055) specific for retinoic acid receptor (RAR) and an agonist (Ro25-7836) specific for retinoid X receptor (RXR) suppressed the AT 1 -R mRNA expression comparable to that with treatment with atRA, suggesting that the RAR/RXR heterodimer mediates the effect of atRA in AT 1 -R downregulation. These results suggest that atRA suppressed AT 1 -R mRNA transcription through new protein synthesis induced by RAR/RXR-dependent transcription. This study provides novel insight into a role of atRA as an important molecule that regulates AT 1 -R gene expression and provides possible mechanisms for the suppression of neointimal formation by atRA.