Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Left ventricular hypertrophy is the primary mechanism by which the heart compensates for a sustained increase in hemodynamic loading. Previous studies have shown that increased load itself is directly linked to hypertrophic growth in terms of both a quantitative increase in cardiac mass and qualitative changes in the cardiac phenotype (Cooper, 1987). However, the signaling mechanisms of hypertrophic cardiac growth are largely unknown. Activation of cell proliferation by hormones and growth factors has been shown to correlate with the intracellular activation of several interacting protein cascades. One of the kinases activated by all mitogens is p70S6 kinase, which leads to phosphorylation of the ribosomal S6 protein and increases the rate of translation of mRNAs containing a polypyrimidine tract. Previous studies have demonstrated P70S6 kinase activation in several cell types after either mitogenic stimulation, mechanical stretch, or integrin receptor engagement. Therefore, p70S6 kinase could play a key role in the load-induced hypertrophic growth process (Laser et al., 1998). Furthermore, the mitogen-activated protein kinases are a superfamily of proline-directed serine/threonine protein kinases and are important mediators of the signal transduction pathway, which is responsible for cellular proliferation. Extracellular signal-regulated kinases (ERKs) are a subgroup of the mitogen-activated protein kinase family and are composed of p42ERK and p44ERK (ERK1/2) (Davis, 1993). Recent reports on cultured cardiac myocytes support the idea that ERKs participate in the mechanism of cardiac hypertrophy and remodeling (Kojima et al., 1994). Moreover, the c-fos gene is the most frequently studied member of the cellular immediate-early genes and has been associated with cellular proliferation, differentiation, and hypertrophy (Verma and Sassone-Corsi, 1987). Therefore, c-fos may play a critical role in myocardial signal transduction.

Angiotensin II (Ang II) induces the early molecular signals of cardiac growth, and stimulates cell growth, myofibrillogenesis, and induction of fetal genes in isolated cardiomyocyte preparations (Baker and Aceto, 1990). Moreover, two main subtypes of Ang II receptor, type 1 (AT1) and type 2, have been identified to date (Matsubara et al., 1994). Most of the known effects of Ang II are mediated through the AT1 receptor, and stimulation of the AT1 receptor produces vasoconstriction, proliferation, and extracellular matrix formation. On the other hand, plasminogen activator inhibitor-1 (PAI-1) is the major inhibitor of tissue and urokinase plasminogen activators and therefore is considered to be an important regulator of fibrinolysis and extracellular matrix turnover (Vassalli et al., 1991). In addition, PAI-1 plays a crucial role in the development of atherosclerosis and neointimal formation after balloon injury. Recent studies have identified an interaction between the renin-angiotensin system (RAS) and the fibrinolytic system (Brown et al., 1998). Ang II may exert an important role in the regulation of circulating and vascular PAI-1 expression. Ang II is a potent stimulator of PAI-1 mRNA and protein expression in both cultured endothelial and vascular smooth muscle cells (SMCs) (Feener et al., 1995). Therefore, these results suggested that activation of the RAS may impair fibrinolysis.

Some studies reported that Rho-kinase, a target protein of small GTP-binding protein Rho, plays crucial roles in various cellular functions and in mediating cellular events such as changes in cell morphology, cell motility, focal adhesions, and cytokinesis (Amano et al., 1997). The possibility that Rho is involved in vascular proliferation and migration is suggested by the involvement of Rho in the growth of nonvascular cells in response to heterotrimeric G protein receptor stimulation and in the migration of endothelial cells in response to mechanical strain or tyrosine kinase growth factors (Santos et al., 1997). Indeed, Rho and the Rho-kinase pathway are involved in DNA synthesis and migration in vascular SMCs of rat aorta (Seasholtz et al., 1999). Moreover, recent studies have demonstrated that cardiomyocyte hypertrophy and myofibrillar assembly are blocked by inhibitory mutants of Rho-kinase, which suggests a role for this Rho effector in cellular growth responses (Hoshijima et al., 1998). Therefore, inhibition of the Rho-kinase pathway may be useful in the treatment of arteriosclerotic cardiovascular diseases. However, the molecular mechanism responsible for the Rho-kinase pathway in Ang II-induced PAI-1 gene expression remains to be determined. Recently, the agent (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632) has been shown to specifically inhibit these Rho-dependent kinases (Uehata et al., 1997). To elucidate the potential cardioprotective effects of a specific Rho-kinase inhibitor and AT1 receptor antagonist, we evaluated the effects of Y-27632 and candesartan on Ang II-induced PAI-1 gene expression and cardiovascular remodeling in Ang II-induced hypertensive rats, and also the contribution of the activation of Rho-kinase and ERK pathway in the left ventricle (LV).

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Animal Model and Experimental Design

All procedures were in accordance with institutional guidelines for animal research. Experiments were performed on 8-week-old male normotensive Wistar-Kyoto rats (Oriental Bioservice Kanto Inc., Ibaraki, Japan). The rats were randomly divided into four groups: 1) a saline-infused group (control group, CON; n = 7), 2) an Ang II-infused group (ANGII-V, n = 7), 3) an Ang II-infused and Y-27632-treated group (ANGII-RHO, n = 7), and 4) an Ang II-infused and candesartan-treated group (ANGII-CAN, n = 7). In all four groups, the rats were anesthetized lightly with ether, and an osmotic minipump (Alzet model 2ML2; Durect Corp., Cupertino, CA) containing Ang II dissolved in saline or saline alone (CON) was implanted subcutaneously. Ang II was continuously infused at 200 ng/kg/min for 14 days. The other osmotic minipump (Alzet model 2 ML2) containing Y-27632 dissolved saline was implanted, and Y-27632 (3 mg/kg/day, subdepressor dose; WelFide Co., Osaka, Japan) was also continuously infused for 14 days. Candesartan (1 mg/kg/day, subdepressor dose; Takeda Chemical Industries, Ltd., Osaka, Japan) was administered orally in a volume of 2 ml/kg as a gum arabic suspension. The body weight of the rats was determined before and at the end of treatment. Systolic blood pressure and heart rate were measured in conscious rats by the tail-cuff method (Muromachi Kikai, model MK-1100, Tokyo, Japan) before treatment and at 1-week intervals thereafter (protocol 1). The numbers of animals and the experimental designs of protocols 2 and 3 were identical to those for protocol 1, above. Rats were housed at a constant temperature (25 ± 1^oC), and were fed standard laboratory rat chow (0.4% sodium content) with free access to drinking water.

Protocol 1

Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Analysis of Rho-Kinase, PAI-1, c-fos, and Endothelial Nitric Oxide Synthase (eNOS) mRNA. After 14 days of treatment, the rats were anesthetized with sodium pentobarbital (50 mg/kg i.p.) and decapitated, and the heart was immediately excised. The LV was carefully separated from the atria and right ventricle, weighed, immediately frozen in liquid nitrogen, and stored at 80°C until extraction of total RNA. The RT-PCR was performed by the standard method with 1 µg of total RNA. First-strand cDNA was synthesized with random primers and Moloney murine leukemia virus reverse transcriptase (Promega). PCR amplification was then performed with synthetic gene-specific primers for Rho-kinase (sense primer, 5'-GCA CAT GTA TGA AAA TGG ATG AAAC-3'; antisense primer, 5'-CAT AAT TTT GCT GTA GGT TCC TAC AAGT-3'), PAI-1 (sense primer, 5'-ATG AGA TCA GTA CTG CGG ACG CCA TCT TTG-3'; antisense primer, 5'-GCA CGG AGA TGG TGC TAC CAT CAG ACT TGT-3'), c-fos (sense primer, 5'-GGG ACA GCC TTT CCT ACT ACC ATT-3'; antisense primer, 5'-CGC AAA AGT CCT GTG TGT TGA-3'), and eNOS (sense primer, 5'-TCC AGT AAC ACA GAC AGT GCA-3'; antisense primer, 5'-CAG GAA GTA AGT GAG AGC-3') using a DNA PCR kit (PerkinElmer Life Sciences, Boston, MA) for 30 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and elongation at 72°C for 1 min. GAPDH was used as a housekeeping gene. Reaction conditions were optimized to obtain reproducible and reliable amplification within the logarithmic phase of the reaction, as determined by preliminary experiments. The reaction was linear to 35 cycles with use of the ethidium bromide detection method. PCR products were separated by electrophoresis on a 2% agarose gel containing ethidium bromide and were visualized by ultraviolet-induced fluorescence. The intensity of each band was quantified using a densitometer. The resulting densities of the Rho-kinase, PAI-1, c-fos, and eNOS bands were expressed relative to the corresponding densities of the GAPDH bands from the same RNA sample (Kobayashi et al., 1999, 2001b,c).

Protocol 2

Western Blot Analysis of RhoA, PAI-1, and eNOS. LV was homogenized (25%, w/v) in 10 mM HEPES buffer, pH 7.4, containing 320 mM sucrose, 1 mM EDTA, 1 mM dithiothreitol, 10 µg/ml leupeptin, and 2 µg/ml aprotinin at 0-4°C with a Polytron homogenizer. Homogenate was centrifuged at 3000g for 5 min at 4°C (eNOS). For PAI-1, the resulting supernatant was then centrifuged and pellets were washed three times with 500 µl of diethyl ether (Motojima et al., 1999). For RhoA, the supernatant was then centrifuged to generate membrane and cytosolic fractions (Sauzeau et al., 2000). Protein concentrations were determined with bovine serum albumin as a standard protein. Equal amounts of protein from membrane and cytosolic fractions (RhoA), the other pellet samples (PAI-1), and the other supernatant samples (eNOS) were loaded in each lane for SDS-polyacrylamide gel electrophoresis using 13% gels. The proteins in the gels were transferred electrophoretically to polyvinylidene difluoride sheets for 1 h at 2 mA/cm². The sheets were immunoblotted with an anti-RhoA, anti-PAI-1, and anti-eNOS antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; and Transduction Laboratories, Lexington, KY) in a buffer containing 10 mM Tris/HCl, pH 7.5, 100 mM NaCl, 0.1% Tween 20, and 5% skim milk, followed by peroxidase-conjugated antimouse and antigoat IgG (Amersham Biosciences, Piscataway, NJ). The RhoA, PAI-1, and eNOS proteins transferred to the sheets were detected using the ECL immunoblotting detection system (Amersham Biosciences) (Kobayashi et al., 2001b,c).

Western Blot Analysis of ERK1/2, P70S6 Kinase, and Myosin Light Chain (MLC) Phosphorylations. Left ventricular ERK1/2, p70S6 kinase, and MLC phosphorylations were measured as described in detail previously (Laser et al., 1998; Kobayashi et al., 2001a). Briefly, by using rabbit polyclonal phospho-ERK1/2, phospho-p70S6 kinase, and goat polyclonal phospho-MLC antibody (New England Biolabs, Beverly, MA; Santa Cruz Biochemicals, Santa Cruz, CA) and anti-total ERK1/2, anti-total p70S6 kinase, and anti-total MLC antibody (New England Biolabs; Santa Cruz Biochemicals) recognizing threonine-phosphorylated forms (active forms) of ERK1/2, p70S6 kinase, and MLC, we measured left ventricular phosphorylated ERK1/2, p70S6 kinase, and MLC proteins with Western blot analysis. Left ventricular protein extracts were boiled for 5 min in Laemmli sample buffer and then electrophoresed by SDS-polyacrylamide gel electrophoresis using 13% gels, and the separated proteins were electrophoretically transferred to Hybond-polyvinylidene difluoride membranes. Complete protein transfer to the membrane was ensured by staining the gels with Coomassie Blue. The membrane was incubated with phospho-specific ERK1/2, p70S6 kinase, and MLC antibody for 1 h at room temperature, washed four times with Tris-buffered saline/Tween 20, and then incubated with horseradish peroxidase-conjugated donkey antirabbit and goat immunoglobulin (Amersham Biosciences).

Protocol 3

Histologic Examination and Evaluation of Cardiovascular Remodeling. Histological examination was studied as described in detail previously (Kobayashi et al., 1999, 2001a,b,c). To assess any thickening of the arterial wall and perivascular fibrosis, transectional images of the area of the total small arteriolar lumen 10⁴ µm² were studied. The wall-to-lumen ratio (the area of the vessel wall divided by the area of the total blood vessel lumen) was determined. The area of fibrosis immediately surrounding the blood vessels was calculated, and perivascular fibrosis was determined as the ratio of the area of fibrosis surrounding the vessel wall to the total area of the vessel. To assess the area of myocardial fibrosis, the area of pathological collagen deposition was measured in the microscopic field of each Masson's trichrome-stained section. The ratio of the total area of fibrosis within the left ventricular myocardium to the total area of the left ventricular myocardium in each heart was calculated and used for analysis. Histopathology on the sections from each rat was carried out by an operator who was blind to the treatment groups.

Statistical Analysis

All results are expressed as the mean ± S.E.M. The mean values were compared among the three or four groups using analysis of variance followed by the Bonferroni test. Differences of p < 0.05 were considered statistically significant. Calculations, including those of derived values, and statistical tests were performed using the appropriate software (StatView-J 4.5; Abacus Concepts, Berkeley, CA) and a Power Macintosh computer system (G4; Apple Computer, Inc., Cupertino, CA).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Systemic Hemodynamics, Body Weight, and Left Ventricular Weight. Systolic blood pressure in ANGII-V, ANGII-RHO, and ANGII-CAN was similar and significantly higher than that in CON (ANGII-V, 174 ± 4; ANGII-RHO, 172 ± 4l; ANGII-CAN, 171 ± 4 versus CON, 134 ± 3 mm Hg, respectively; p < 0.01). Heart rate was similar in CON and ANGII-V (325 ± 9 versus 331 ± 10 bpm), and was not changed by the administration of Y-27632 (337 ± 9 bpm versus ANGII-V) or candesartan (340 ± 9 bpm versus ANGII-V). Body weight was also similar among the four groups (CON, 258 ± 5; ANGII-V, 251 ± 7; ANGII-RHO, 255 ± 6; and ANGII-CAN, 253 ± 7 g). The left ventricular weight was significantly greater in ANGII-V than in CON (2.54 ± 0.05 versus 2.01 ± 0.03 mg/g, p < 0.01), and was significantly less in ANGII-RHO and ANGII-CAN than in ANGII-V (ANGII-RHO, 2.20 ± 0.04; ANGII-CAN, 2.22 ± 0.04 mg/g versus ANGII-V, respectively; p < 0.01).

Effects of Y-27632 and Candesartan on RhoA and Rho-Kinase Expression and Activity in Ang II-Induced Hypertensive Rats. The levels of RhoA in membrane fraction of LV were 3.7-fold (p < 0.01) larger in ANGII-V than in CON, and were lower in ANGII-RHO and ANGII-CAN (65 and 63%, respectively; p < 0.01) than in ANGII-V (Fig. 1A). Expressions of Rho-kinase mRNA were 4.3-fold (p < 0.01) larger in ANGII-V than in CON, and were lower in ANGII-RHO and ANGII-CAN (65 and 67%, respectively; p < 0.01) than in ANGII-V (Fig. 1B). In addition, to quantify the activity of Rho-kinase in hypertensive heart, we performed Western blot analysis for phosphorylated MLC in the LV. Left ventricular phospho-MLC activity was 4.2-fold (p < 0.01) larger in ANGII-V than in CON, and was lower in ANGII-RHO and ANGII-CAN (74 and 76%, respectively; p < 0.01) than in ANGII-V (Fig. 1C).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Effects of Y-27632 and candesartan treatment on RhoA protein (A), Rho-kinase mRNA (B), and phospho-MLC (C). Top panels are representative of typical RT-PCR and Western blot bands. Bottom panels show percentage of control of RhoA (A) and phospho-MLC (C), and the mean densities of the Rho-kinase (B) bands in relation to GAPDH. Lane 1 refers to control rats (CON); lane 2, to angiotensin II-induced hypertensive rats treated with the vehicle (ANGII-V); lane 3, to angiotensin II-induced hypertensive rats treated with Y-27632 (ANGII-RHO); and lane 4, to angiotensin II-induced hypertensive rats treated with candesartan (ANGII-CAN). Values are expressed as means ± S.E.M., n = 7 per group. , p < 0.01 versus CON; , p < 0.01 versus ANGII-V.

Effect of Y-27632 and Candesartan on Ang II-Induced PAI-1 and c-fos Expression. Left ventricular PAI-1 mRNA levels were 3.2-fold (p < 0.01) larger in ANGII-V than in CON, and the Ang II-induced PAI-1 gene expression was inhibited by Y-27632 and candesartan (60 and 58%, respectively; p < 0.01) (Fig. 2A). The levels of PAI-1 protein in the LV were 3.4-fold (p < 0.01) larger in ANGII-V than in CON, and were lower in ANGII-RHO and ANGII-CAN (59 and 58%, respectively; p < 0.01) than in ANGII-V (Fig. 2B). The levels of c-fos mRNA were 3.1-fold (p < 0.01) larger in ANGII-V than in CON, and the Ang II-induced c-fos gene expression was inhibited by Y-27632 and candesartan (61 and 58%, respectively; p < 0.01) (Fig. 2C).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Effects of Y-27632 or candesartan treatment on PAI-1 mRNA (A), PAI-1 protein (B) and c-fos (C) mRNA expression. Top panels are representative typical RT-PCR and Western blot bands. Bottom panels show the mean densities of the PAI-1 (A) and c-fos (C) bands in relation to GAPDH, and percent of control of PAI-1 (B). Lane 1 refers to control rats (CON), lane 2, to angiotensin II-induced hypertensive rats treated with the vehicle (ANGII-V), lane 3, to angiotensin II-induced hypertensive rats treated with Y-27632 (ANGII-RHO), and lane 4, to angiotensin II-induced hypertensive rats treated with candesartan (ANGII-CAN). Values are expressed as means ± S.E.M., n = 7 per group. , p < 0.01 versus CON; , p < 0.01 versus ANGII-V.

The Relationship between Rho/Rho-Kinase and ERK/p70S6 Kinase Pathways in Ang II-Induced Hypertensive Rats. To evaluate the relationship between the Rho/Rho-kinase and ERK/p70S6 kinase pathways, we examined whether the Rho/Rho-kinase pathway was involved in ERK1/2 and p70S6 kinase activities in hypertensive heart. Left ventricular phospho-ERK1/2 activities were significantly higher in ANGII-V than in CON, and the Ang II-induced phosphorylation of ERK1/2 activation was not inhibited by Y-27632 but by candesartan (Fig. 3A). Moreover, in a similar manner, cardiac phospho-p70S6 kinase activity was significantly increased in ANGII-V compared with CON, and the Ang II-stimulated phospho-p70S6 kinase activation was not inhibited by Y-27632 but by candesartan (Fig. 3B).

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Effects of Y-27632 or candesartan treatment on phospho-ERK1/2 (A) and phospho-p70S6 kinase (B) activities. Top panels are representative of typical Western blots of phospho- and total ERK1/2, and phospho- and total p70S6 kinase in the LV. Bottom panels show percentage of control of left ventricular phospho-ERK1/2 and phospho-p70S6 kinase activities. Lane 1 refers to control rats (CON); lane 2, to angiotensin II-induced hypertensive rats treated with the vehicle (ANGII-V); lane 3, to angiotensin II-induced hypertensive rats treated with Y-27632 (ANGII-RHO); and lane 4, to angiotensin II-induced hypertensive rats treated with candesartan (ANGII-CAN). Values are expressed as means ± S.E.M., n = 7 per group. , p < 0.01 versus CON; , p < 0.05, , p < 0.01 versus ANGII-V; , p < 0.05, , p < 0.01 versus ANGII-RHO.

Cardiovascular Remodeling. The wall-to-lumen ratio was increased in ANGII-V compared with CON but was significantly decreased by Y-27632 and candesartan treatment (Figs. 4 and 5A). The degree of perivascular fibrosis was significantly greater in ANGII-V than in CON, and was also significantly decreased by Y-27632 and candesartan treatment (Figs. 4 and 5B). Compared with CON, myocardial fibrosis was significantly greater in ANGII-V, but it was significantly less in ANGII-RHO and ANGII-CAN than in ANGII-V (Fig. 5C).

View larger version (109K): [in this window] [in a new window]   Fig. 4.   Micrographs of small coronary arteries with Masson's trichrome stain for control rats (A), angiotensin II-induced hypertensive rats treated with the vehicle (B), angiotensin II-induced hypertensive rats treated with Y-27632 (C), and angiotensin II-induced hypertensive rats treated with candesartan (D). Bar, 100 µm.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Effects of Y-27632 or candesartan on cardiovascular remodeling in angiotensin II-induced hypertensive rats. Wall-to-lumen ratio (A), perivascular fibrosis (B), and myocardial fibrosis (C) were measured histopathologically. CON, control rats; ANGII-V, angiotensin II-induced hypertensive rats treated with vehicle; ANGII-RHO, angiotensin II-induced hypertensive rats treated with Y-27632; ANGII-CAN, angiotensin II-induced hypertensive rats treated with candesartan. Values are expressed as means ± S.E.M., n = 7 per group. , p < 0.05, , p < 0.01 versus CON. , p < 0.01 versus ANGII-V.

Effect of Y-27632 on eNOS mRNA and Protein Levels. To evaluate the mechanisms of cardioprotective effect of inhibiting the Rho-kinase pathway, expression of eNOS mRNA and protein was measured. Left ventricular eNOS mRNA levels were 61% (p < 0.05) lower in ANGII-V than in CON, and were 4.6-fold (p < 0.01) larger in ANGII-RHO than in ANGII-V (Fig. 6A). The levels of eNOS protein in the LV were 59% (p < 0.05) lower in ANGII-V than in CON, and were 5.9-fold (p < 0.01) larger in ANGII-RHO than in ANGII-V (Fig. 6B).

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Effects of Y-27632 treatment on eNOS mRNA (A) and eNOS protein (B). Top panels are representative of typical RT-PCR and Western blot bands. Bottom panels show the mean densities of eNOS mRNA bands in relation to GAPDH (A), and percentage of control of eNOS protein (B). Lane 1 refers to control rats (CON); lane 2, to angiotensin II-induced hypertensive rats treated with vehicle (ANGII-V); and lane 3, to angiotensin II-induced hypertensive rats treated with Y-27632 (ANGII-RHO). Values are expressed as means ± S.E.M., n = 7 per group. , p < 0.05 versus CON; , p < 0.01 versus ANGII-V.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In the present study, we demonstrated that Rho and Rho-kinase play a key role in Ang II-induced hypertensive rats and cardiovascular remodeling. Ang II-induced PAI-1 gene expression through the AT1 receptor and the Rho and Rho-kinase pathways is critical in Ang II-induced PAI-1 gene up-regulation. Rho and Rho-kinase were not involved in Ang II-induced phosphorylation of ERK1/2 and p70S6 kinase activation. Rho-kinase was involved in Ang II-induced expression of the c-fos gene. These results suggested that differential activation of Rho-kinase and ERK pathways may play a critical role in Ang II-induced PAI-1 gene expression, and up-regulation of Rho-kinase plays a key role in the pathogenesis of Ang II-induced hypertensive rats.

PAI-1 is an important regulator of fibrinolysis, and up-regulation of PAI-1 levels in plasma are a risk factor for arterial thrombotic disease (Vassalli et al., 1991). In addition, recent studies have shown an increase of local PAI-1 mRNA expression, predominantly in SMCs, within human atherosclerotic lesions (Christ et al., 1997). These observations suggest a relationship between PAI-1 expression and cell proliferation and support the concept that high expression of PAI-1 may correlate with the progression of atherosclerosis (Sironi et al., 2001). In this study, we showed that Rho-kinase inhibitor Y-27632 and AT1 receptor antagonist candesartan effectively inhibited Ang II-induced PAI-1 gene expression. Ang II has been shown to increase PAI-1 expression in SMCs isolated from rat aorta, through an interaction with the AT1 receptor subtype (Feener et al., 1995). Sironi et al. (2001) indicated that AT1 receptor antagonist completely prevented the enhancing effects of Ang II on PAI-1 in human arterial SMCs. Moreover, Chen et al. (2000) reported that vascular PAI-1 gene expression was elevated in spontaneously hypertensive rat, and AT1 receptor antagonism could reduce elevated vascular PAI-1 gene. They concluded that the RAS influences vascular PAI-1 expression in rats primarily through the AT1 pathway. Furthermore, some studies have demonstrated that activation of RhoA enhances activator protein-1 (AP-1) and its transcriptional activity, and these factors are responsible for Ang II-induced PAI-1 up-regulation via Rho-kinase activation (Chang et al., 1998). Takeda et al. (2001) showed that activation of the Rho-kinase pathway plays a pivotal role in PAI-1 gene up-regulation by Ang II. Taken together, these results suggested that AT1 receptor and the Rho-kinase pathway may play a critical role in Ang II-induced PAI-1 gene up-regulation.

In the present study, we have shown that the Rho-kinase pathway plays a critical role in Ang II-induced cardiac hypertrophy and cardiovascular remodeling by using the specific Rho-kinase inhibitor Y-27632 in vivo. The Rho and Rho-kinase pathway plays an important role in regulation of vascular SMC contraction and other cellular functions such as proliferation and migration. In vitro studies by Yamakawa et al. (2000) examined the effects of Y-27632 on Ang II-induced leucine uptake in vascular SMCs. They showed that pretreatment of the cells with Y-27632 dose dependently suppressed the leucine incorporation induced by Ang II. Kuwahara et al. (1999) evaluated the Rho/ROCK pathway in endothelin-1 (ET-1)-induced hypertrophic signals in cardiac myocytes. They indicated that Y-27632 significantly suppressed ET-1-induced hypertrophic response: augmentation of natriuretic peptide gene expression, increase in protein synthesis and cell size, and myofibrillar reorganization. More recently, Sauzeau et al. (2001) have reported that human urotensin II-induced vascular SMC proliferation is inhibited by Y-27632 or TAT-C3, a RhoA inhibitor, indicating that RhoA and Rho-kinase mediate the stimulation of vascular SMC growth. Moreover, in vivo studies by Eto et al. (2000) showed that Rho-kinase was functionally up-regulated at the balloon-injured site of the porcine femoral artery, and gene transfer of dominant-negative Rho-kinase significantly suppressed the development of neointimal formation after balloon injury. They concluded that Rho-kinase was involved in the pathogenesis of neointimal formation after balloon injury in vivo. Recently, Mukai et al. (2001) examined the role of Rho-kinase in functional and structural alterations of hypertensive blood vessels in spontaneously hypertensive rats. They have concluded that up-regulation of Rho-kinase plays a key role in the pathogenesis of hypertensive vascular disease. These results suggested that Rho and Rho-kinase might be involved in Ang II-induced cardiovascular remodeling.

Activation of ERK1/2 and p70S6 kinase has been reported to be closely related to protein synthesis in vascular SMCs. In vitro studies show that ERKs are rapidly activated by various growth factors and vasoactive hormones such as Ang II (Davis, 1993). In addition, Hamaguchi et al. (1998) showed that Ang II-induced hypertension caused the activation of glomerular ERK, leading to the activation of AP-1, and suggested that ERK signaling cascades, via the activation of AP-1, might be implicated in the development of hypertension-induced glomerular injury in vivo. Therefore, ERK1/2 is regarded as a critical mediator for cell growth and vascular hypertrophy. However, the relation between these kinases and Rho-kinase has not been clearly identified. We examined whether Rho-kinase might be involved in Ang II-induced ERK1/2 and p70S6 kinase activation. Numaguchi et al. (1999) reported that the Rho and Rho-kinase pathway regulated mechanical stretch-induced ERK1/2 activation in vascular SMCs. Moreover, Hill et al. (1995) demonstrated that Rho was involved in lysophosphatidic acid-induced ERK activation in NIH 3T3 cells. Furthermore, Matrougui et al. (2001) indicated that Rho-kinase inhibition decreased Ang II-induced ERK1/2 activation in isolated, intact mesenteric resistance arteries. In contrast, in the present study, we showed that Y-27632 had no effect on Ang II-stimulated phospho-ERK1/2 and phospho-p70S6 kinase activities, but candesartan inhibited these phosphorylated kinases. Aikawa et al. (1999) indicated that Rho was not involved in Ang II-induced ERK activation in cardiac myocytes. Takeda et al. (2001) and Funakoshi et al. (2001) also demonstrated that inhibition of Rho-kinase Y-27632 did not affect Ang II-induced ERK activation in vascular SMCs. Besides, Yamakawa et al. (2000) reported that Y-27632 had no effect on the ERK1/2 and p70S6 kinase phosphorylations induced by Ang II in vascular SMCs. These results suggested that Rho and Rho-kinase regulate Ang II-induced PAI-1 expression mediated by a pathway different from ERK1/2 or p70S6 kinase phosphorylations. Thus, the Rho-kinase and ERK pathways may be independent of each other in the signaling of Ang II. However, further investigations are required to clarify these differences in signaling pathways.

In this study, we showed that Rho and Rho-kinase were involved in Ang II-induced expression of the c-fos gene, and also that candesartan inhibited this gene expression. Aikawa et al. (1999) examined the role of Rho proteins in mechanical stress-induced gene expression in cardiac myocytes. They showed that Rho proteins were essential for mechanical stress-induced expression of c-fos gene. Yamakawa et al. (2000) indicated that Rho-kinase is partially involved in Ang II-induced c-fos gene expression in vascular SMCs. Moreover, Ueyama et al. (1997) reported that activated RhoA stimulated c-fos gene expression in myocardial cells. These results suggested that Ang II-induced expression of the c-fos gene might be mediated through activation of the Rho-kinase and ERK1/2 pathway.

Uehata et al. (1997) have demonstrated that the agent Y-27632 has been shown to specifically inhibit Rho-dependent kinases (K_i = 0.14 µM for p160ROCK: >100 times selectivity versus protein kinase C, cAMP-dependent protein kinase, and MLC kinase). A new pyridine derivative, Y-27632, selectively inhibits smooth-muscle contraction by inhibiting the Ca²⁺ sensitization mechanism. This compound inhibited smooth-muscle contraction both in vitro and in vivo, as well as the formation of stress fibers and focal adhesions induced by p160ROCK in cultured cells. Therefore, Y-27632 is a valuable tool for investigating the functions of p160ROCK in vivo and its pathophysiological implications. As well as modulating smooth-muscle contraction, the Rho-kinase pathway may help to regulate integrin-mediated cell adhesion and motility, which could be critical in processes such as tumor cell metastasis and immunoactivation. Thus, Y-27632 should be useful for investigating the role of p160ROCK in these processes and may be clinically important (Uehata et al., 1997).

In the present study, we demonstrated that the cardioprotective effects of candesartan and Y-27632 were independent of blood pressure as neither agent at the doses used blocked Ang II-induced hypertension. With regard to candesartan, we have previously shown that coronary microvascular remodeling in normotensive and Ang II-induced hypertensive rats was significantly ameliorated by a subdepressor dose of candesartan (1 mg/kg/day), which may be mediated, at least in part, by an increase in local eNOS mRNA and protein expression, and nitric oxide synthase activity in the LV (Kobayashi et al., 2001b). In addition, we have reported that up-regulated preproET-1, endothelin A receptor, and platelet-derived growth factor (PDGF) A-chain mRNA expression in Ang II-induced hypertensive rats were significantly decreased by a subdepressor dose of candesartan (1 mg/kg/day). Moreover, after 2 weeks of treatment, candesartan effectively improved left ventricular hypertrophy and cardiovascular structural changes, and decreased type I collagen mRNA expression, possibly due to a decrease in preproET-1, endothelin A receptor, and PDGF A-chain mRNA expression in the LV (Hara et al., 2001). Therefore, in the present study, these results suggested that cardiovascular remodeling in Ang II-induced hypertensive rats was significantly ameliorated by a subdepressor dose of candesartan, which may be in part mediated by an increase in eNOS expression and a decrease in ET-1 and PDGF A-chain expression in the LV.

The mechanisms of the beneficial effect of inhibiting Rho-kinase are unknown. We demonstrated that eNOS expression was up-regulated by inhibiting Rho-kinase. Laufs and Liao (1998) reported that the up-regulation of eNOS expression by hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitor is mediated by inhibition of Rho GTPase. They concluded that Rho negatively regulated eNOS expression and that HMG-CoA reductase inhibitors up-regulated eNOS expression by blocking Rho. These results suggest that the production of eNOS expression through the inhibition of Rho-kinase may play a critical role in the protective effect of cardiovascular remodeling.