A single amino acid substitution in the 126-kDa protein of pepper mild mottle virus controls replication and systemic movement into upper non-inoculated leaves of bell pepper plants.

Previously, we generated attenuated variants of pepper mild mottle virus by replacing residue 649 in the 126-kDa replicase protein with various amino acids. Here, we examined the biological properties of the 16 variants that caused either mild mosaic or no mosaic. All but one (A649N) of the mild-mosaic-inducing strains replicated at higher levels in pepper plants and systemically moved at higher rates into the upper non-inoculated leaves than the no-mosaic strains. C1421, previously selected for practical use, not only caused mild symptoms but also had an especially high replication rate in pepper plants and spread more efficiently into the upper non-inoculated leaves.

A single residue in the 126-kDa protein of pepper mild mottle virus controls the severity of symptoms on infected green bell pepper plants.

Infectious cDNA clones originally derived from a mild strain of Pepper mild mottle virus were constructed by replacing residue 649, a critical point for attenuation of this virus, with all possible amino acids. All clones were infectious to pepper plants and induced a variety of symptoms, including no visible symptoms. The results of this study showed that a single amino acid mutation at residue 649 could control the function of the 126- and 183-kDa proteins, replicases with multiple roles in the life cycle of this virus.

Integration of mutations responsible for the attenuated phenotype of Pepper mild mottle virus strains results in a symptomless cross-protecting strain.

An enhanced attenuated strain of Pepper mild mottle virus (PMMoV) was constructed by incorporating mutations that affect viral attenuation from three reported attenuated strains of PMMoV, which causes serious economic losses in the production of green pepper in Japan. The new strain caused no symptoms on pepper plants and protected them from infection by a wild-type strain. The mutations responsible for viral attenuation were located in the intervening region (IR) of the 126-kDa/183-kDa proteins. The mutations had synergistic effects in terms of the attenuation of symptoms and decreased the accumulation of the viral coat protein in infected pepper plants. In this paper, we propose an efficient method for the improvement of attenuated viruses by reverse genetics in plant viruses.

A single amino acid substitution in 126-kDa protein of Pepper mild mottle virus associates with symptom attenuation in pepper; the complete nucleotide sequence of an attenuated strain, C-1421.

The complete nucleotide sequence of an attenuated Pepper mild mottle virus (PMMoV C-1421) RNA genome has been determined. There were two differences from the type isolate in Japan (PMMoV-J). The mutations were located in the middle of the 126-kDa protein (126 K) gene; one mutation influenced amino acid substitution at 649th Val to Ala (V649A), and the other was silent. The analyses using the reverse genetic system of PMMoV-J revealed that symptom attenuation on pepper related to V649A. Accumulations of 126 K and coat protein (CP) in V649A mutant-infected pepper were lower than those of PMMoV-J in immunoblotting. These results suggest that V649A substitution in 126 K affects the accumulation of 126 K leading to a limitation of CP accumulation.

Downregulation of angiotensin II type 1 receptor by hydrophobic 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors in vascular smooth muscle cells.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors, so-called statins, reduce the relative risk of a major coronary event by lowering the serum cholesterol level. In addition, statins may confer beneficial effects by cholesterol-lowering independent mechanisms, which are incompletely characterized. Because angiotensin II (Ang II) plays crucial roles in the pathogenesis of cardiovascular diseases, we examined the effect of statins on the expression of the Ang II type 1 receptor (AT(1)-R) in cultured vascular smooth muscle cells (VSMCs). Cerivastatin and fluvastatin reduced the AT(1)-R mRNA and the AT(1)-R protein levels; however, pravastatin lacked this effect. Cerivastatin and fluvastatin suppressed the AT(1)-R promoter activity measured by luciferase assay but did not affect AT(1)-R mRNA stability, suggesting that the suppression occurs at the transcriptional level. Coincubation of VSMCs with mevalonate or geranylgeranyl pyrophosphate but not with farnesyl pyrophosphate reversed the cerivastatin-induced AT(1)-R downregulation. Overexpression of dominant-negative Rho A also suppressed AT(1)-R mRNA expression. Treatment with cerivastatin for 24 hours reduced the calcium response of VSMCs to Ang II. Taken together, statins downregulate AT(1)-R expression through a mevalonate-dependent, geranylgeranyl pyrophosphate-dependent, and Rho A-dependent manner and attenuate the biological function of Ang II. Downregulation of AT(1)-R may contribute to the cholesterol-independent beneficial effect of statins on the cardiovascular system.

Thrombin induces interleukin-6 expression through the cAMP response element in vascular smooth muscle cells.

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 -228 and -150 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.

cAMP response element-binding protein mediates thrombin-induced proliferation of vascular smooth muscle cells.

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 [(3)H]thymidine and [(3)H]leucine. These results suggest that CREB-dependent gene transcription plays a critical role in thrombin-induced proliferation and hypertrophy of VSMCs. Transactivation of the epidermal growth factor receptor and 2 mitogen-activated protein kinase pathways are involved in this process. CREB may be a novel transcription factor mediating the vascular remodeling process induced by thrombin.

15-Deoxy-delta 12,14-prostaglandin J2 and thiazolidinediones activate the MEK/ERK pathway through phosphatidylinositol 3-kinase in vascular smooth muscle cells.

Peroxisome proliferator-activated receptor (PPAR) gamma belongs to the nuclear receptor superfamily of ligand-dependent transcription factors. Recent results have shown that the ligands for nuclear receptors have rapid effects so called "nongenomic" effects, which are observed within minutes after stimulation. We examined whether 15-deoxy-Delta(12,14)-prostaglandin J(2) (15-d-PGJ2) had rapid effects on cultured vascular smooth muscle cells. Phosphorylation of ERK and c-fos mRNA expression were determined by Western and Northern blot analyses, respectively. PPAR gamma agonists 15-d-PGJ2 and thiazolidinediones such as pioglitazone and troglitazone elicited rapid activation of ERK within 15 min and induced c-fos mRNA expression within 30 min, whereas the PPAR alpha agonist bezafibrate failed to activate ERK. 15-d-PGJ2-induced expression of c-fos mRNA was blocked by PD98059 or U0126, two ERK kinase inhibitors, suggesting that the MEK/ERK pathway mediates 15-d-PGJ2-induced c-fos gene expression. Furthermore, pretreatment with wortmannin, an inhibitor of phosphatidylinositol 3 (PI3)-kinase, inhibited 15-d-PGJ2-induced ERK activation and c-fos mRNA expression, suggesting that PI3-kinase is involved in the process. An electrophoretic mobility shift assay showed that 15-d-PGJ2 enhanced AP-1 binding activity to AP-1 consensus sequence in a time-dependent manner. 15-d-PGJ2 increased thymidine incorporation in a PI3-kinase-dependent manner. Taken together, our findings show that 15-d-PGJ2 and thiazolidinediones activate the MEK/ERK pathway through PI3-kinase and lead to c-fos mRNA expression and DNA synthesis. These findings indicate a novel regulatory mechanism of gene expression by 15-d-PGJ2 and thiazolidinediones.

Solid-state conformation of a hybrid tripeptide between beta-amino acid; 8-aminocyclooct-4-enecarboxylic acid and 2-aminoisobutyric acid.

An eight-membered cyclic beta-amino acid, 8-aminocyclooct-4-enecarboxylic acid, was designed as a conformationally restricted non-proteinogenic amino acid. A hybrid tripeptide containing this eight-membered cyclic beta-amino acid and 2-aminoisobutyric acids was synthesized by conventional solution methods. The conformation of the tripeptide was studied using X-ray analysis and was shown to form an eleven-membered hydrogen-bonded turn (3(11)-helical structure) in the solid state.

Rho-kinase mediates angiotensin II-induced monocyte chemoattractant protein-1 expression in rat vascular smooth muscle cells.

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. To examine the role of Rho-kinase in Ang II-induced MCP-1 expression, we used adenovirus-mediated overexpression of the dominant negative mutant of Rho-kinase (AdDNRhoK) or Y-27632, a specific inhibitor of Rho-kinase. Both AdDNRhoK and Y-27632 strongly inhibited Ang II-induced MCP-1 expression. Although inhibition of extracellular signal-regulated protein kinase (ERK) by PD 098,059 also inhibited Ang II-induced MCP-1 expression, Y-27632 did not affect Ang II-induced activation of ERK. These results indicate that Rho-kinase plays a critical role in Ang II-induced MCP-1 production independent of ERK. The Rho-Rho-kinase pathway may be a novel target for the inhibition of Ang II signaling and the treatment of atherosclerosis.

Angiotensin II type 2 receptor is essential for left ventricular hypertrophy and cardiac fibrosis in chronic angiotensin II-induced hypertension.

BACKGROUND: The roles of angiotensin II (Ang II) in the regulation of heart function under normal and pathological conditions have been well documented. Although 2 types of Ang II receptor (AT(1) and AT(2)) are found in various proportions, most studies have focused on AT(1)-coupled events. In the present study, we examined the hypothesis that signaling by AT(2) is important to the development of left ventricular hypertrophy and cardiac fibrosis by Ang II infusion in mice lacking the AT(2) gene (Agtr2-/Y). METHODS AND RESULTS: Male Agtr2-/Y and age-matched wild-type (WT) mice were treated long-term with Ang II, infused at a rate of 4.2 ng. kg(-1). min(-1) for 3 weeks. Ang II elevated systolic blood pressure to comparable levels in Agtr2-/Y and WT mice. WT mice developed prominent concentric cardiac hypertrophy, prominent fibrosis, and impaired diastolic relaxation after Ang II infusion. In contrast, there was no cardiac hypertrophy in Agtr2-/Y mice. Agtr2-/Y mice, however, did not show signs of heart failure or impairment of ventricular relaxation and only negligible fibrosis after Ang II infusion. The absence of fibrosis may be a clue to the absence of impairment in ventricular relaxation and account for the normal left ventricular systolic and diastolic performances in Agtr2-/Y mice. CONCLUSIONS: Chronic loss of AT(2) by gene targeting abolished left ventricular hypertrophy and cardiac fibrosis in mice with Ang II-induced hypertension.

Critical role of Rho-kinase and MEK/ERK pathways for angiotensin II-induced plasminogen activator inhibitor type-1 gene expression.

Plasminogen activator inhibitor type-1 (PAI-1) plays an integral role not only in the regulation of fibrinolytic activity but also in the pathogenesis of atherosclerosis and hypertension. We investigated the signaling pathways of angiotensin II (Ang II) leading to PAI-1 gene expression. Ang II increased the PAI-1 mRNA and protein levels in a time- and dose-dependent manner through the Ang II type 1 receptor in vascular smooth muscle cells. PAI-1 gene promoter activity measured by luciferase assay was significantly increased by Ang II. PAI-1 mRNA stability was also increased by Ang II. Ang II-induced PAI-1 mRNA upregulation was inhibited by BAPTA-AM, genistein, and AG1478, suggesting that intracellular calcium, tyrosine kinase, and epidermal growth factor receptor transactivation are involved. Furthermore, PD98059, an inhibitor of extracellular signal-regulated kinase (ERK) kinase (MEK), almost completely suppressed Ang II-induced PAI-1 upregulation. Adenovirus-mediated overexpression of the dominant-negative form of Rho-kinase or Y27632, a Rho-kinase inhibitor, also completely prevented PAI-1 induction by Ang II without affecting Ang II-induced ERK activation. These data suggest that activation of MEK/ERK and Rho-kinase pathways plays a pivotal role in PAI-1 gene upregulation by Ang II. The Rho-kinase pathway may be a novel target to inhibit Ang II signaling, and its inhibition may be useful in the treatment of hypertension as well as atherosclerosis.

Electroosmosis injection of blood serum into biocompatible microcapillary chip fabricated on quartz plate.

A chip which allows the detection of various human health markers from a trace amount of blood has been studied. As a goal, a microcapillary with a 30 x 30 microm cross-section was fabricated using all-dry etching technologies on a 2 x 2 cm SiO2 chip. The coating of the biocompatible 2-methacryloyloxyethylphosphorylcholine (MPC) polymer on the inner quartz wall of the microcapillary demonstrated a sufficiently long adsorption suppression of proteins in the serum on the quartz surface, while rapid stopping occurred for serum injected into the microcapillary with a bare quartz surface. The latter rapid stopping corresponded well to fast electroosmosis flow due to the negatively increasing zeta-potential by the adsorption of proteins on the quartz surface. The electroosmosis pump arranged a downstream of the microcapillary was also developed to inject serum into it. As a preliminary application, a given concentration-standard solution was injected into the ion-sensitive field-effect transistor (ISFET) embedded in the chip, employing the electroosmosis pump arranged downstream of the sensor position. Hence, the pH and Na+ and K+ cation concentrations were measured.

Reactive oxygen species-mediated homologous downregulation of angiotensin II type 1 receptor mRNA by angiotensin II.

Recent studies suggest a crucial role of reactive oxygen species (ROS) for the signaling of angiotensin (Ang) II through Ang II type 1 receptor (AT(1)-R). However, the role of ROS in the regulation of AT(1)-R expression has not been explored. In this study, we examined the effect of an antioxidant on the homologous downregulation of AT(1)-R by Ang II. Ang II (10(-6) mol/L) decreased AT(1)-R mRNA with a peak suppression at 6 hours of stimulation in rat aortic vascular smooth muscle cells. Preincubation of vascular smooth muscle cells with N:-acetylcysteine (NAC), a potent antioxidant, almost completely inhibited the Ang II-induced downregulation of AT(1)-R mRNA. The effect of NAC was due to stabilization of the AT(1)-R mRNA that was destabilized by Ang II. The Ang II-induced AT(1)-R mRNA downregulation was also blocked by PD98059, an extracellular signal-regulated protein kinase (ERK) kinase inhibitor. Ang II-induced ERK activation was inhibited by NAC as well as by PD98059. Exogenous H(2)O(2) also suppressed AT(1)-R mRNA. These results suggest that the production of ROS and the activation of ERK are critical for the downregulation of AT(1)-R mRNA. The generation of ROS through stimulation of AT(1)-R not only mediates signaling of Ang II but also may play a crucial role in the adaptation process of AT(1)-R to the sustained stimulation of Ang II.

Relationship between regional severity of emphysema and coronary heart disease.

We analyzed the relationship between regional severity of emphysema, which was evaluated by three-dimensional fractal analysis (3D-FA) of Technegas SPECT images, and coronary heart disease (CHD). For 22 patients with emphysema who underwent Technegas SPECT, we followed up CHD events. The follow-up period was 5.4+/-0.5 (mean +/- SD) years. We defined the upper-lung fractal dimension (U-FD) and lower-lung fractal dimension (L-FD) obtained with 3D-FA of Technegas SPECT images as the regional severity of emphysema. FD became greater with the progression of emphysematous change. During the follow-up period, CHD events occurred in 6 (27%) of the 22 patients. The ratio of U-FD to L-FD for patients with CHD events (0.87+/-0.22) was significantly smaller than for patients without CHD events (1.52+/-0.38) (p = 0.0015). These findings suggest that severer emphysema in the lower lung indicates a higher risk of CHD than that in the upper lung.

Pain threshold, learning and formation of brain edema in mice lacking the angiotensin II type 2 receptor.

The main biological role of angiotensin II type 2 receptor (AT2) has not been established. We made use of targeted disruption of the mouse AT2 gene to examine the functional role of the AT2 receptor in the central nervous system (CNS). We have previously shown that AT2-deficient mice displayed anxiety-like behavior in comparisons with wild-type mice. In the present study, we analyzed the pain threshold, learning behavior and brain edema formation using the tail-flick test, the tail-pinch test, the passive avoidance task and cold injury, respectively. In the passive avoidance task and cold injury, no differences were found between wild-type mice and AT2-deficient mice. In contrast, the pain threshold was significantly lower in AT2-deficient mice, compared with findings in wild-type mice. The immunohistochemical distribution of beta-endorphin in the brain was analyzed quantitatively in AT2-deficient mice and wild-type mice, using microphotometry. The fluorescence intensity of beta-endorphin in the arcuate nucleus of the medial basal hypothalamus (ARC) was significantly lower in AT2-deficient mice, compared with findings in wild-type mice. We found that the AT2 receptor does not influence learning behavior and brain edema formation. As AT2-deficient mice have increased sensitivity to pain and decreased levels of brain beta-endorphin, AT2 receptors may perhaps mediate regulation of the pain threshold.

Peroxisome proliferator-activated receptor gamma activators downregulate angiotensin II type 1 receptor in vascular smooth muscle cells.

BACKGROUND: Peroxisome proliferator-activated receptor gamma (PPARgamma) 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 PPARgamma 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 PPARgamma ligand 15-deoxy-Delta(12,14)-prostaglandin J(2), as well as Tro, reduced the AT(1)-R mRNA expression and the AT(1)-R protein level. The PPARgamma activators also reduced the calcium response of VSMCs to Ang II. PPARgamma 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: PPARgamma 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 PPARgamma activators.

Quantitative analysis of technegas SPECT: evaluation of regional severity of emphysema.

UNLABELLED: The purpose of this study was to quantify the regional severity of emphysema by 3-dimensional fractal analysis of technegas (99mTc-carbon particle radioaerosol) SPECT images. METHODS: Technegas SPECT was performed on 22 patients with emphysema. The lungs were delineated using 4 cutoff levels (15%, 20%, 25%, and 30% of the maximal pixel radioactivity), and the total number of pixels was measured in the areas surrounded by the contours obtained with each cutoff level. We calculated fractal dimensions from the relationship between the total number of pixels and cutoff levels transformed into logarithms. Fractal dimension for total or regional lung was defined as the severity of emphysema. RESULTS: Total lung fractal dimension (T-FD), upper lung fractal dimension (U-FD), and lower lung fractal dimension (L-FD) for patients with emphysema were 1.84 +/- 0.46 (mean +/-SD), 2.22 +/- 0.61, and 1.77 +/- 0.49, respectively. U-FD was significantly greater than was L-FD. Patients with the ratio of U-FD to L-FD of <1.16 had a significantly greater percentage forced vital capacity (FVC) than did patients with the ratio of >1.16. Patients with an L-FD of <1.8 had a significantly greater forced expiratory volume in 1 s (FEV1)/FVC than did patients with that of >1.8. No significant difference was found between patient groups stratified by U-FD. CONCLUSION: The regional severity of emphysema was well shown by these fractal dimensions.

Expression of monocyte chemoattractant protein-1 by nonenzymatically glycated albumin (Amadori adducts) in vascular smooth muscle cells.

The biological effects of Amadori adducts that are early nonenzymatically glycated protein on vascular cells were poorly defined. We examined the effect of glycated serum albumin (GA) on the expression of monocyte chemoattractant protein-1(MCP-1) that is an important chemokine recruiting monocyte to blood vessel. GA increased MCP-1 mRNA expression with a peak after 3 h of stimulation. The induction of MCP-1 by GA was dose-dependent. The MCP-1 mRNA expression by GA was completely inhibited by PD98059 and genistein that inhibit mitogen activated protein (MAP) kinase kinase and tyrosine kinase, respectively. N-Acetylcysteine, a potent antioxidant, also suppressed the GA-induced MCP-1 expression. These results suggest that GA induces production of reactive oxygen species and activates tyrosine kinase and MAP kinase in VSMC. Activation of these signals results in MCP-1 expression. GA-induced MCP-1 expression may be one of the mechanisms by which the diabetic patients suffer from accelerated atherosclerosis.

Downregulation of angiotensin II type 1 receptor by all-trans retinoic acid in vascular smooth muscle cells.

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 micromol/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.