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. Author manuscript; available in PMC: 2009 Jan 9.
Published in final edited form as: J Am Coll Cardiol. 2007 Jan 26;49(6):698–705. doi: 10.1016/j.jacc.2006.06.082

Roles of Rho-Associated Kinase and Oxidative Stress in the Pathogenesis of Aortic Stiffness

Kensuke Noma *, Chikara Goto *, Kenji Nishioka *, Daisuke Jitsuiki *, Takashi Umemura *, Keiko Ueda †, Masashi Kimura †, Keigo Nakagawa †, Tetsuya Oshima ‡, Kazuaki Chayama †, Masao Yoshizumi *, James K Liao §, Yukihito Higashi *
PMCID: PMC2615568  NIHMSID: NIHMS84974  PMID: 17291936

Abstract

Objectives

The purpose of this study was to determine the relationship between Rho-associated kinase (ROCK) activity and aortic stiffness in humans.

Background

Epidemiologic studies have shown that there is a relationship between aortic stiffness and cardiovascular complications. Recent evidence suggests that ROCK plays an important role in the process of atherosclerosis.

Methods

We evaluated the forearm blood flow (FBF) response to sodium nitroprusside (SNP), a nitric oxide donor, acetylcholine (ACh), an endothelium-dependent vasodilator, and fasudil, a specific ROCK inhibitor, in 51 healthy male subjects (mean age 45.6 ± 3.0 years). The FBF was measured by using a strain-gauge plethysmograph. Carotidfemoral pulse wave velocity (cf-PWV) was measured to assess the aortic stiffness using a pulse wave velocimeter.

Results

Intra-arterial infusion of SNP alone, ACh alone, or fasudil alone and after coinfusion of NG-monomethyl-L-arginine (L-NMMA), a nitric-oxide synthase inhibitor, significantly increased FBF in a dose-dependent manner (p < 0.01). Multivariate analysis showed that age and number of pack-years smoked were independent predictors of ROCK activity before or after co-infusion of L-NMMA (p < 0.01) and that age and ROCK activity before or after co-infusion of L-NMMA were independent predictors of cf-PWV (p < 0.01). The concentration of serum malondialdehyde-modified low-density lipoprotein, an index of oxidative stress, was significantly correlated with ROCK activity before and after co-infusion of L-NMMA and cf-PWV (p < 0.01).

Conclusions

These findings suggest that aging and accumulating smoking habit, which might induce excessive oxidative stress, are involved in ROCK activity in the vasculature, leading to an increase in aortic stiffness in humans.

The small guanosine triphosphatase (GTPase) Rho works as a switch and plays an important role in various cellular physiologic functions, including actomyosin-based cellular processes such as cell adhesion, migration, motility, cytokinesis, and contraction, all of which may be involved in the pathogenesis of atherosclerosis (1). There is growing evidence that Rho-associated kinase (ROCK) (also known as Rho-kinase), the immediate downstream target of the small GTP-binding protein Rho, contributes to endothelial dysfunction and vascular disease (2-6). Indeed, recent clinical evidence has demonstrated that ROCK is significantly activated in patients with coronary vasospasm (7), hypertension (8), and stable-effort angina (9) and even in current smoking subjects (10,11). Rho-kinase, therefore, is becoming a new therapeutic target in cardiovascular disease. Rho-kinase physiologically plays a key role in vasoconstriction. It activates myosin light chain kinase by phosphorylation of the myosin-binding subunit in myosin light chain phosphatase, leading to contraction of vascular smooth muscle cells (VSMC) (12,13). Thus, the vasoconstriction mediated by ROCK is dependent on Ca2+ sensitization but independent of Ca2+ concentration. Earlier studies have known that vascular dysfunction, including endothelial and VSMC dysfunction, is associated with cardiovascular risk factors such as aging, smoking habit, and oxidative stress (14,15), but little is known about the underlying correlations of ROCK with cardiovascular risk factors. In addition, there is no clinical evidence of a relationship between ROCK and oxidative stress, although recent studies have revealed a relationship between endothelial dysfunction and ROCK (3,4,11,16) and between endothelial dysfunction and oxidative stress (17,18). Several recent studies have provided evidence that reactive oxygen species activate the Rho/ROCK pathway (19,20). Indeed, we previously showed that there is a significant relationship between endothelial dysfunction and increased ROCK activity in young current smokers, which might be considered as a human model of excess oxidative stress compared with young nonsmokers (11). Taken together, these findings indicate that reactive oxygen species may play an important role in activation of ROCK in humans.

Epidemiologic and clinical studies have shown that impaired aortic stiffness, which can be assessed noninvasively by measurement of pulse wave velocity (PWV), is an independent marker to estimate subjects with cardiovascular disease (21,22). The PWV depends on arterial wall structure, mainly collagen and elastin, and arterial function (23). The elastic properties of the aorta and central arteries are the major determinants of systemic arterial impedance, and PWV measured along the aortic and aortoiliac pathway is the most clinically relevant.

To determine the roles of ROCK and oxidative stress in the pathogenesis of impaired aortic stiffness, we evaluated whether oxidative stress is related to ROCK activity and subsequently to aortic stiffness in humans.

Methods

Subjects

We studied 51 healthy male subjects (mean age 45.6 ± 3.0 years). Subjects with a history of hypertension, hypercholesterolemia, or diabetes mellitus were excluded. Normal blood pressure was defined as systolic blood pressure of <140 mm Hg and diastolic blood pressure of <85 mm Hg. The results of physical and routine laboratory examinations of the subjects were normal. None of the subjects were taking oral antioxidant vitamins or vasoactive drugs. Current smokers were defined as any who had smoked at least 1 pack-year. One pack-year was defined as 20 cigarettes per day for 1 year. All of the smokers (35.2 ± 5.2 pack-years) had a current smoking history of more than 5 years and abstained from smoking for at least 3 h before the forearm blood flow (FBF) measurements. We defined nonsmokers as those who had never smoked. The study protocol was approved by the Ethical Committee of Hiroshima University Graduate School of Biomedical Sciences. Informed consent for participation in the study was obtained from all subjects.

Procedures

Forearm vascular response to sodium nitroprusside (SNP) (Maluishi Pharmaceutical Co.) alone, acetylcholine (ACh) (Daiichi Pharmaceutical Co.) alone, and fasudil (Asahi Chemical Industries) alone and after coinfusion of NG-monomethyl-L-arginine (L-NMMA) (Sigma Chemical Co.), were evaluated. The study began at 8:30 am with the subjects in the fasting condition. A 23-gauge polyethylene catheter (Hokkow Co.) was inserted into the left brachial artery for the infusion of each drug and for the recording of arterial pressure with an AP-641G pressure transducer (Nihon Kohden Co.) under local anesthesia (1% lidocaine). Another catheter was inserted into the left deep antecubital vein to obtain blood samples.

After 30 min in the supine position, we measured basal FBF and arterial blood pressure. Then forearm vascular response to SNP, a direct vasodilator of VSMCs, ACh, an endothelium-dependent vasodilator, and fasudil, a specific ROCK inhibitor, on forearm hemodynamics were measured. The SNP (0.75, 1.5, or 3.0 μg/min), ACh (3.75 or 7.5 μg/min), and fasudil (3, 10, or 30 μg/min) were infused intra-arterially for 5 min at each dose. The FBF was measured during the last 2 min of the infusion. The infusions of SNP, ACh, and fasudil were carried out in a randomized fashion. Each study proceeded after the FBF returned to baseline.

After a 30-min rest period, L-NMMA, an inhibitor of nitric oxide synthase, was infused intra-arterially at a dose of 8 μmol/min for 5 min, and fasudil was administered 5 min after the initiation of L-NMMA.

To evaluate the relationship among ROCK activity, oxidative stress, and aortic stiffness, we studied 35 healthy male subjects (mean age 45.0 ± 3.6 years), whose prespecified entry criteria are identical to that described in the preceding. Number of pack-years smoked in smokers was 29.0 ± 6.6 pack-years. The PWV was measured in the supine position after 15 minutes of bed rest, and then forearm vasodilative responses to SNP alone, ACh alone, and fasudil alone and after co-infusion of L-NMMA, were evaluated in a manner identical to that described in the preceding text.

Measurement of FBF

The FBF was measured with a mercury-filled Silastic strain-gauge plethysmography (EC-5R, D. E. Hokanson) as previously described (11,18).

Measurement of PWV

Aortic compliance was assessed noninvasively on the basis of Doppler ultrasound measurements of PWV along the descending thoracoabdominal aorta as previously published and validated (24). Briefly, carotid-femoral PWV (cf-PWV), an index of arterial stiffness, was determined by 2 pressure sensors, placed on the right femoral and left carotid arteries to record each pulse wave simultaneously, and the time lag (t) between the notches of the 2 waves, using a pulse wave velocimeter (Form PWV/ABI, model BP-203RPE, Colin Co.). The distance (D) between the 2 recording sensors was calculated automatically from the value of individual height. The PWV value was calculated as PWV = D/t. The PWV was measured for 5 consecutive pulses, and averages were used for analysis. The observer was blind to the form of the examination.

Analytical methods

Routine chemical methods were used to determine serum concentrations of total cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides, glucose, and insulin. The serum concentration of malondialdehyde-modified low-density lipoprotein (MDA-LDL) was assayed by ELISA (antiMDA-LDL antibody, SRL Co.). Fasting concentrations of insulin and glucose were used to determine homeostatic model of assessment (HOMA) parameters of insulin resistance using a program based on the HOMA algorithm (insulin resistance = insulin/22.5e-In glucose), as previously described (25).

Statistical analysis

Results are presented as mean ± SEM. Values of p < 0.05 were considered to indicate statistical significance. Comparisons of time-course curves of variables during the infusion of SNP alone, ACh alone, and fasudil alone and after co-infusion of L-NMMA, were analyzed by analysis of variance (ANOVA) for repeated measures with Bonferroni correction to baseline. The ROCK activity was expressed as the percentage in the ratio of fasudil-stimulated maximal FBF relative to the immediately preceding basal FBF. Spearman rank correlation was used to compare ROCK activity before or after co-infusion of L-NMMA with age, body mass index (BMI), systolic blood pressure, diastolic blood pressure, heart rate, serum concentration of total cholesterol, HDL cholesterol, triglycerides, mean fasting glucose, insulin, MDA-LDL, and number of pack-years smoked. The analysis was also used to compare the ratio of SNP-stimulated maximal FBF to basal FBF or ACh-stimulated maximal FBF to basal FBF (endothelial function) with the variables and to compare the PWV with the variables in which endothelial function and ROCK activity before or after L-NMMA were added. Multivariate analysis using multiple stepwise regression was performed to determine the significant correlation of ROCK activity before or after co-infusion of L-NMMA and of PWV with variables which showed a p value of <0.1 in Spearman correlation analysis. Multivariate analysis was performed with the Statistical Analysis System program package (SAS Institute, Cary, North Carolina). The data were analyzed using the software package StatView V and Super ANOVA (Abacus Concepts).

Results

Clinical characteristics

The baseline clinical characteristics of the subjects are summarized in Table 1.

Table 1. Baseline Clinical Characteristics in the Subjects.

Variable Subjects (n = 51)
Age (yrs) 45.6 ± 3.0
Body mass index (kg/m2) 23.5 ± 0.4
Systolic blood pressure (mm Hg) 123.6 ± 1.4
Diastolic blood pressure (mm Hg) 65.5 ± 1.4
Heart rate (beats/min) 62.5 ± 1.1
Total cholesterol (mmol/l) 4.70 ± 0.10
HDL cholesterol (mmol/l) 1.33 ± 0.06
Triglyceride (mmol/l) 1.03 ± 0.03
Mean fasting glucose (mmol/l) 5.1 ± 0.1
Serum insulin (pmol/l) 73.9 ± 16.6
Insulin resistance (HOMA index) 3.17 ± 0.81
Current smoker 27
Number of pack-yrs smoked in smokers 35.2 ± 5.2
Basal FBF (ml/min/100 ml tissue) 7.2 ± 0.4
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All results are presented as mean ± SEM.

FBF = forearm blood flow; HDL = high-density lipoprotein; HOMA = homeostatic model assessment.

Effects of FBF responses to SNP, ACh, and fasudil

Intra-arterial infusion of SNP significantly increased FBF in a dose-dependent manner by ANOVA for repeated measurements (6.1 ± 0.3 ml/min to 31.1 ± 1.4 ml/min per 100 ml tissue; p < 0.01) (Fig. 1). Intra-arterial infusion of ACh also significantly increased FBF in a dose-dependent manner by ANOVA for repeated measurements (7.7 ± 0.6 ml/min to 16.6 ± 1.5 ml/min per 100 ml tissue; p < 0.01) (Fig. 2). Intra-arterial infusion of fasudil significantly increased FBF in a dose-dependent manner alone and after co-infusion of L-NMMA by ANOVA for repeated measurements (7.0 ± 0.4 ml/min to 12.3 ± 0.6 ml/min per 100 ml tissue, 5.2 ± 0.4 ml/min vs. 10.3 ± 0.7 ml/min per 100 ml tissue, respectively; p < 0.01) (Fig. 3). No significant change was observed in arterial blood pressure or heart rate during intra-arterial infusion of any drugs.

Figure 1. FBF Response to SNP.

Figure 1

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Results are presented as mean ± SEM. The probability value refers to a comparison of time course curves by analysis of variance for repeated measurements. *p < 0.01 vs. basal forearm blood flow (FBF) assessed by Bonferroni correction. SNP = sodium nitroprusside.

Figure 2. FBF Response to ACh.

Figure 2

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Results are presented as mean ± SEM. The probability value refers to a comparison of time course curves by analysis of variance for repeated measurements. *p < 0.05; †p < 0.01 vs. basal forearm blood flow (FBF) assessed by Bonferroni correction. ACh = acetylcholine.

Figure 3. FBF Response to Fasudil Alone and After Co-Infusion of L-NMMA.

Figure 3

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Results are presented as mean ± SEM. The probability value refers to a comparison of time course curves by analysis of variance for repeated measurements in each group. *p < 0.01 vs. basal forearm blood flow (FBF) of fasudil alone; †p < 0.01 vs. basal FBF after co-infusion of NG-Monomethyl-L-arginine (L-NMMA) assessed by Bonferroni correction.

Correlations with ROCK activity before and after L-NMMA

Rho-associated kinase activity was significantly correlated with age (r = 0.64; p < 0.001), systolic blood pressure (r = 0.44; p < 0.01), serum concentration of total cholesterol (r = 0.41; p < 0.01), and number of pack-years smoked (r = 0.52; p < 0.001). The ROCK activity after co-infusion of L-NMMA was significantly correlated with age (r = 0.64; p < 0.01), systolic blood pressure (r = 0.40; p < 0.01), serum concentration of total cholesterol (r = 0.47; p < 0.01), and number of pack-years smoked (r = 0.49; p < 0.01). The ROCK activity before or after co-infusion of L-NMMA was not correlated with other parameters. The correlations between ROCK activity before or after co-infusion of L-NMMA and variables are summarized in Table 2. Stepwise multiple regression analysis was carried out to identify the independent predictors of ROCK activity before or after co-infusion of L-NMMA. For multiple regression analysis, variables showing a p value of <0.1 in Spearman correlation analysis were selected; age, systolic blood pressure, serum concentration of total cholesterol, and number of pack-years smoked were entered as candidates for independent variables. Stepwise multiple regression analysis revealed that age (standardized r = 0.36) and number of pack-years smoked (standardized r = 0.39) were independent predictors of ROCK activity (multiple R2 = 0.43; p < 0.01) and that age (standardized r = 0.34) and number of pack-years smoked (standardized r = 0.35) were independent predictors of ROCK activity after coinfusion of L-NMMA (multiple R2 = 0.37; p < 0.01). The serum concentration of MDA-LDL was significantly correlated with ROCK activity before and after co-infusion of L-NMMA (r = 0.57 and r = 0.51, respectively; p < 0.01).

Table 2. Correlations Between ROCK Activity Before and After L-NMMA and Variables.

ROCK Activity Before L-NMMA
 ROCK Activity After L-NMMA
Variable Coefficient p Value Coefficient p Value
Age (yrs) 0.636 <0.001 0.635 <0.001
Body mass index (kg/m2) -0.019 0.892 -0.007 0.961
Systolic blood pressure (mm Hg) 0.437 0.002 0.396 0.007
Diastolic blood pressure (mm Hg) -0.093 0.516 -0.134 0.358
Heart rate (beats/min) -0.089 0.533 -0.131 0.368
Total cholesterol (mmol/l) 0.414 0.004 0.471 0.001
HDL cholesterol (mmol/l) -0.157 0.277 -0.156 0.290
Triglyceride (mmol/l) 0.167 0.248 0.089 0.547
Mean fasting glucose (mmol/l) 0.097 0.555 -0.048 0.778
Serum insulin (pmol/l) 0.066 0.677 0.087 0.590
Insulin resistance (HOMA index) 0.033 0.852 0.026 0.888
Number of pack-yrs smoked 0.515 <0.001 0.487 <0.001
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L-NMMA = NG-monomethyl-L-arginine; ROCK Rho-associated kinase; other abbreviations as in Table 1.

Correlations with maximal FBF response to SNP

There was no variable that was significantly correlated with the ratio of SNP-stimulated maximal FBF to basal FBF (Table 3). The serum concentration of MDA-LDL was also not significantly correlated with FBF response to SNP (r = -0.04; p = 0.81).

Table 3. Correlations Between the Ratio of SNP-Stimulated Maximal FBF to Basal FBF and Variables.

Variables Coefficient p Value
Age (yrs) -0.127 0.372
Body mass index (kg/m2) -0.049 0.730
Systolic blood pressure (mm Hg) 0.107 0.453
Diastolic blood pressure (mm Hg) 0.040 0.780
Heart rate (beats/min) -0.086 0.546
Total cholesterol (mmol/l) -0.120 0.407
HDL cholesterol (mmol/l) 0.005 0.972
Triglyceride (mmol/l) 0.147 0.308
Mean fasting glucose (mmol/l) 0.049 0.768
Serum insulin (pmol/l) -0.045 0.776
Insulin resistance (HOMA index) -0.044 0.801
Number of pack-yrs smoked 0.021 0.886
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SNP = sodium nitroprusside; other abbreviations as in Table 1.

Correlations with endothelial function

Endothelial function was significant correlated with age (r = -0.55; p < 0.01), serum concentration of total cholesterol (r = -0.67; p < 0.01), and number of pack-years smoked (r = -0.59; p < 0.01) (Table 4). The serum concentration of MDA-LDL was also significantly correlated with endothelial function (r = -0.64; p < 0.01).

Table 4. Correlations Between Endothelial Function and Variables.

Variables Coefficient p Value
Age (yrs) -0.548 0.003
Body mass index (kg/m2) -0.020 0.915
Systolic blood pressure (mm Hg) -0.313 0.092
Diastolic blood pressure (mm Hg) 0.140 0.451
Heart rate (beats/min) 0.218 0.241
Total cholesterol (mmol/l) -0.671 <0.001
HDL cholesterol (mmol/l) 0.071 0.709
Triglyceride (mmol/l) -0.074 0.694
Mean fasting glucose (mmol/l) 0.281 0.187
Serum insulin (pmol/l) 0.179 0.353
Insulin resistance (HOMA index) 0.346 0.113
Number of pack-yrs smoked -0.588 0.002
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Abbreviations as in Table 1.

Correlations with cf-PWV

The Cf-PWV was significantly correlated with age (r = 0.88, p < 0.01), systolic blood pressure (r = 0.42; p < 0.05), serum concentration of total cholesterol (r = 0.47; p < 0.01), number of pack-years smoked (r = 0.38; p < 0.05), endothelial function (r = -0.54; p < 0.01), ROCK activity (r = 0.67; p < 0.01), and ROCK activity after co-infusion of L-NMMA (r = 0.75; p < 0.01). The ratio of SNP-stimulated maximal FBF to basal FBF was not significantly correlated with cf-PWV. The cf-PWV was not correlated with other parameters. The correlations between cf-PWV and variables are summarized in Table 5. For multiple regression analysis, variables showing a p value of <0.1 in Spearman correlation analysis were selected; age, systolic blood pressure, serum concentration of total cholesterol, number of pack-years smoked, endothelial function, and ROCK activity before or after L-NMMA,were entered as candidates for independent variables. Stepwise multiple regression analysis revealed that age (standardized r = 0.71) and ROCK activity (standardized r = 0.27) were independent predictors of cf-PWV (multiple R2 = 0.79; p < 0.01) and that age (standardized r = 0.51) and ROCK activity after coinfusion of L-NMMA (standardized r = 0.54) were independent predictors of cf-PWV (multiple R2 = 0.91; p < 0.01). In addition, the serum concentration of MDA-LDL was significantly correlated with cf-PWV (r = 0.57; p < 0.01).

Table 5. Correlations Between PWV and Variables.

Variables Coefficient p Value
Age (yrs) 0.882 <0.001
Body mass index (kg/m2) 0.025 0.884
Systolic blood pressure (mm Hg) 0.418 0.015
Diastolic blood pressure (mm Hg) 0.211 0.218
Heart rate (beats/min) -0.141 0.411
Total cholesterol (mmol/l) 0.472 0.006
HDL cholesterol (mmol/l) -0.085 0.620
Triglyceride (mmol/l) 0.133 0.437
Mean fasting glucose (mmol/l) -0.069 0.695
Serum insulin (pmol/l) -0.060 0.729
Insulin resistance (HOMA index) -0.145 0.421
Number of pack-yrs smoked 0.377 0.028
The ratio of SNP-stimulated maximal FBF to basal FBF (%) -0.155 0.373
Endothelial function (%) -0.543 0.009
ROCK activity (%) 0.670 <0.001
ROCK activity after coinfusion of L-NMMA (%) 0.746 <0.001
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Abbreviations as in Tables 1, 2, and 3.

Discussion

In the present study, there were significant relationships between ROCK activity and age, systolic blood pressure, serum concentration of total cholesterol, and number of pack-years smoked in healthy male subjects. In multivariate analysis, age and number of pack-years smoked were independent predictors of ROCK activity among the candidates that were correlated with ROCK activity. In addition, the concentration of serum MDA-LDL, one of the established markers of oxidative stress, was correlated with ROCK activity. To the best of our knowledge, this is the first study to provide clinical evidence revealing significant involvement of ROCK activity with age, accumulating current smoking habit, and oxidative stress in humans.

Recently, increasing evidence has indicated that ROCK is significantly associated with the regulation of not only endothelial nitric oxide synthase (eNOS) expression but also eNOS phosphorylation, both of which are important mechanisms for regulating endothelial function and subsequent cardiovascular injury (2-4,16). We have also demonstrated a significant relationship between endothelial dysfunction and increased ROCK activity in current smokers (11). The results of the present study show that aging and cigarette smoking are involved in an increase in ROCK activity, which might be partly explained by the significant correlation between ROCK and endothelial function. Our results are supported by several recent studies showing that aging is significantly related to activation of the Rho/ROCK pathway (26,27). Those findings suggest that activation of ROCK is involved in several aspects of the atherosclerotic process, including endothelial dysfunction.

Fasudil, which competes with ATP for binding to ATP-dependent kinase domains, has recently been shown to be a potent and specific inhibitor of ROCK (5,6,28). Rho-kinase activity in humans has been investigated in several previous studies using fasudil (8,29). Masumoto et al. (7). demonstrated that ROCK activity, evaluated by the vasodilative response to fasudil, is increased at the segment of coronary vasospasm in patients with vasospastic angina. In addition, we evaluated ROCK activity after co-infusion of L-NMMA to assess ROCK activity in the forearm vasculature. Although it is not clear whether ROCK activity before or after co-infusion of L-NMMA is appropriate for assessing ROCK activity, ROCK activity after co-infusion of L-NMMA may be appropriate for precise assessment of ROCK activity because it may avoid the contribution of endogenous nitric oxide to ROCK in VSMC. Interestingly, we could not find a significant correlation of forearm vasodilatory response to SNP with the variables in this study, which is in accordance with previous evidence (8,18). This discrepancy between the vasodilative effects of fasudil and SNP may be due to the different pharmacologic mechanisms of the drugs, dependency on Ca2+ sensitivity, or Ca2+ concentration in VSMC.

One of the novel findings in the present study is that ROCK activity, but not endothelial function, is one of the independent predictors of increased PWV, indicating that ROCK is significantly involved in the pathogenesis of aortic stiffness. Aortic stiffness has been shown to be closely associated with cardiovascular risks (22,24,30) and to be a significant predictor of cardiovascular morbidity and mortality in subjects with a smoking habit, hypertension, endstage renal disease, and aging (21,31,32). Of great interest are the results of a recent study by Willum-Hansen et al. (33) showing that PWV predicted a composite of cardiovascular outcomes over a median follow-up period of 9.4 years beyond 24-h mean arterial pressure and other traditional risk factors such as gender, age, body mass index, current smoking habit, and alcohol intake in middle-aged and elderly individuals, results that are significant from the point of view of clinical benefit. According to previous studies, ROCK plays an important role in atherosclerotic processes, especially in VSMC (5,6,12,13,28), indicating that ROCK modulates VSMC function via several kinds of mechanisms, including regulation of Ca2+ sensitivity, which may explain the discrepancy in the results of the present study showing that ROCK activity but not endothelial function is an independent predictor of PWV.

Oxidative stress is known to be crucial for the development of cardiovascular disease and subsequent mortality (15,34). In the present study, we measured the concentration of serum MDA-LDL to evaluate the contribution of oxidative stress to ROCK activity and aortic stiffness. Thus, the significant correlations of ROCK activity with age and number of pack-years smoked may be evoked partly through excess oxidative stress. Indeed, several investigators have demonstrated a possible correlation of ROCK with oxidative stress in in vitro and in vivo studies (20). Consequently, it is feasible that oxidative stress is significantly correlated with ROCK activity and further aortic stiffness in the present study. Collectively, these findings support our hypothesis that activation of ROCK in VSMC leads to impaired aortic stiffness, although we could not determine from the results of the present study whether increased ROCK activity causes oxidative stress or whether oxidative stress caused by various variables such as aging and accumulating smoking habit leads to increased ROCK activity. Furthermore, endothelial dysfunction, which is significantly related not only to excess oxidative stress but also to activated ROCK, may also be an important mechanism of impairment of aortic stiffness. Although the precise mechanism remains to be determined, ROCK plays a critical role in the modulation of aortic stiffness through a pathway in which oxidative stress is involved.

Study limitations

Although, we obtained the striking finding that aortic stiffness significantly correlates with ROCK and oxidative stress, several limitations remain in the present study. It has been shown that creatinine clearance is negatively associated with PWV in subjects with moderate reduction of creatinine clearance (35). Although serum concentrations of creatinine in all subjects in this study were normal (less than 106 μmol/l), additional data on the correlation of creatinine clearance with ROCK or PWV might make the results more plausible. In addition, recent studies have revealed significant relationships between aortic stiffness and several candidates such as serum high-sensitivity C-reactive protein (hs-CRP) and criteria for the diagnosis of the metabolic syndrome (36-38). Indeed, it has been shown that CRP activates ROCK, leading to plasminogen activator inhibitor 1 expression and atherothrombogenesis in vitro. Those finding suggest that serum level of hs-CRP may correlate with ROCK activity and may cause the elevation of PWV in humans (39). In the present study, waist circumference was not measured. The BMI was measured in all subjects instead of waist circumference, and BMI was <30 kg/m2 in all subjects. Accordingly, our results might not have been greatly influenced by the lack of measurement of waist circumference. It is thought that many disorders of biologic and physiologic factors are related to impaired aortic stiffness. Therefore, further investigations are required to clarify the precise mechanism underlying impairment of aortic stiffness.

Conclusions

We have demonstrated that ROCK activity in the forearm vasculature is significantly associated with age and number of pack-years smoked and that increased cf-PWV is significantly related to age and ROCK activity. In addition, excess oxidative stress is significantly correlated with increased ROCK activity and PWV. These findings suggest that excessive oxidative stress might be involved in increased ROCK activity in the vasculature, leading to impaired aortic stiffness, and that not only oxidative stress but also ROCK might be vital therapeutic targets for cardiovascular protection.

Acknowledgments

The authors thank the Research Foundation for Community Medicine. The authors also thank Naotsugu Oyama for his considerable advice.

Supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan and a Japan Heart Foundation grant for research on hypertension and vascular metabolism.

Abbreviations and Acronyms

ACh

acetylcholine

cf-PWV

carotid-femoral pulse wave velocity

FBF

forearm blood flow

L-NMMA

NG-monomethyl-L-arginine

MDA-LDL

malondialdehyde-modified low-density lipoprotein

PWV

pulse wave velocity

ROCK

Rho-associated kinase

SNP

sodium nitroprusside

VSMC

vascular smooth muscle cells

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