Abstract
Objective
Cardiovascular diseases are associated with chronic activation of Rho-associated kinases (ROCKs) and endothelial dysfunction. Both increased ROCK activity and endothelial dysfunction are thought to be closely associated with conventional cardiovascular risk factors. The purpose of this study was to determine the relationship between ROCK activity, endothelial function and cardiovascular risk factors.
Methods and Results
We evaluated ROCK activity in peripheral leukocytes by Western blot analysis and flow-mediated vasodilation by ultrasonography in 242 men who had no cardiovascular or cerebrovascular diseases (mean age, 40±10 yr; range, 20 to 73 yr). ROCK activity was defined as the ratio of phospho myosin-binding subunit on myosin light chain phosphatase to total myosin-binding subunit. Univariate regression analysis revealed that leukocyte ROCK activity significantly correlated with body mass index (r=0.29, P=0.003), systolic blood pressure (r=0.25, P=0.01), low-density lipoprotein cholesterol level (r=0.21, P=0.04), and Framingham risk factor score, a cumulative cardiovascular risk index for heart attack, (r=0.31, P<0.001) and that flow-mediated vasodilation significantly correlated with age (r=−0.23, P=0.02), body mass index (r=0.19, P=0.05), systolic blood pressure (r=−0.22, P=0.03), total cholesterol level (r=−0.21, P=0.04), low-density lipoprotein cholesterol level (r=−0.22, P=0.04), glucose level (r=−0.20, P=0.04), and Framingham risk factor score (r=−0.37, P<0.001). There was a significant correlation between leukocyte ROCK activity and flow-mediated vasodilation (r=−0.41, P<0.001). Multivariate analysis revealed that flow-mediated vasodilation was an independent predictor of leukocyte ROCK activity.
Conclusions
These findings suggest cumulative cardiovascular risk may enhance ROCK activity and endothelial dysfunction, leading to progression of cardiovascular diseases and outcomes.
Keywords: Rho-associated kinases, endothelial function, Framingham risk factor score
Introduction
Rho-associated kinases (ROCKs), one of the first downstream targets of the small GTP-binding protein Rho A, play a pivotal role in the regulation of vascular smooth muscle contraction, endothelial function, and many cellular functions.1-5 Several lines of evidence have shown that ROCK activity is enhanced in patients with angina pectoris, vasospastic angina, pulmonary hypertension, heart failure, and stroke.6-10 Increased ROCK activity may play a critical role in the pathogenesis and progression of several cardiovascular diseases.
The vascular endothelium is involved in the release of various vasodilators, including nitric oxide, prostacyclin and endothelium-derived hyperpolarizing factor, as well as vasoconstrictors.11,12 Nitric oxide plays an important role in the regulation of vascular tone, inhibition of platelet aggregation, and suppression of smooth muscle cell proliferation. Impaired endothelium-dependent vasodilation has been found in the forearm, coronary, and renal vasculature in patients with hypertension,13-17 hypercholesterolemia,18 diabetes mellitus,19 and coronary artery diseases.20,21 Endothelial dysfunction is the initial step in the pathogenesis of atherosclerosis, resulting in adverse cardiovascular and cerebrovascular outcomes.22-24 Previous studies have shown that activation of the RhoA/ROCK pathway impaired nitric oxide bioavailability through downregulation of endothelial nitric oxide synthase mRNA stability and inhibition of endothelial nitric oxide synthase protein phosphorylation at Ser 1177 via the Akt/PI3K pathway, suggesting that there is an interaction between ROCK activity and endothelial function.25,26
To evaluate the relationships between ROCK activity, and conventional cardiovascular risk factors and endothelial function, we measured the leukocyte ROCK activity and flow-mediated vasodilation in healthy men and those with cardiovascular risk factors but without established cardiovascular or cerebrovascular diseases.
Methods
Subjects
A total of 242 men (mean age, 43±10 yr; age range, 20 to 73 yr) were recruited from people who underwent health screening examinations at Ten-noz Clinic and Hiroshima University Hospital. Hypertension was defined as systolic blood pressure of more than 140 mm Hg and/or diastolic blood pressure of more than 90 mm Hg, in a sitting position, on at least three different occasions. Patients with secondary forms of hypertension were excluded on the basis of complete history, physical examination, radiological and ultrasound examinations, urinalysis, plasma renin activity, plasma aldosterone and norepinephrine concentrations, serum creatinine, potassium, calcium, and free thyroxine concentrations, and 24-hour urinary excretion of 17-hydroxycorticosteroids, 17-ketogenic steroids, and vanillymandelic acid. Normotension was defined as systolic blood pressure of less than 140 mm Hg and diastolic blood pressure 90 mm Hg. Diabetes was defined according to the American Diabetes Association27 and/or a previous diagnosis of diabetes. Dyslipidemia was defined according to the third report of the National Cholesterol Education Program.28 None of the patients had a history of cardiovascular or cerebrovascular disease, liver disease, or renal disease. The healthy subjects had no history of serious disease. All subjects took no medication for at least 12 weeks before the study. Women were excluded from this study because of potential confounding factors, including menstrual cycle. Framingham risk factor score was calculated by points of risk factors: age, total cholesterol level, high-density lipoprotein cholesterol level, systolic blood pressure, and smoking status.29 The ethical committees of our institutes approved the study protocol. Written informed consent for participation in the study was obtained from all of the subjects.
Subjects fasted the previous night for at least 12 hours. After remaining in the supine position for 30 minutes, basal ROCK activity and fasting serum concentrations of total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, creatinine, insulin, glucose, and electrolytes were measured.
Measurement of ROCK activity
ROCK activity was assayed in peripheral blood leukocytes as the amount of phospho-Thr853 in the myosin-binding subunit (p-MBS) of myosin light chain phosphatase. Blood was collected at room temperature in heparinized tubes (20 U/mL). After adding an equal volume of 2% dextran, the sample was kept at room temperature for 30 min. The supernatant was spun at 1450 rpm. for 10 min. Red blood cells in the resulting cell pellet were lysed with the addition of water and spun at 1450 rpm for 10 min after the addition of Hank’s balanced salt solution (Hyclone, Logan, UT, USA). The resulting leukocyte pellet was resuspended in medium 199 (Sigma Chemical Co., Saint Louis, Missouri, USA) and counted using a hematocytometer. Cells were fixed in 10% trichloroacetic acid and 10mmol/L dichlorodiphenyltrichloroethane. After centrifugation, the cell pellets were stored at 80°C for western blot analysis. Cells pellets were dissolved in 10 μL of 1 mol/L Tris base and then mixed with 100 μL of extraction buffer (8 mol/L urea, 2% sodium dodecyl sulfate, 5% sucrose, and 5% 2-mercaptoethanol). Equal amounts of cell extracts were subjected to 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. NIH 3T3 cell lysates were used as a positive control and to standardize the results of western blot analyses from several membranes. After serum starvation for 20 hours, confluent cells were stimulated with 10 μmol/L lysophosphatidic acid for 10 minutes and then subsequently fixed and harvested in 10% trichloroacetic acid and 10 mmol/L dichlorodiphenyltrichloroethane. Following centrifugation at 1450 rpm for 10 minutes at 4°C, precipitates were dissolved in 10 μL of 1 mol/L Tris base and mixed with 100 μL of extraction buffer. An equal volume of positive control cell lysate was used for each gel. Membranes were incubated with rabbit anti–phospho-specific Thr853–MBS polyclonal antibody (Biosource Invitrogen, Carlsbad, California, USA) or rabbit anti-MBS polyclonal antibody (Covance Laboratories, Evansville, Indiana, USA), or antiactin monoclonal antibody (Sigma). Bands were visualized using the ECL system (Amersham-Pharmacia Co., London, UK). Images were captured using Adobe Photoshop, and the band intensities were quantified using National Institutes of Health Image 1.61. ROCK activity was expressed as the ratio of p–MBS in each sample to p–MBS in each positive control divided by total MBS (t-MBS) in each sample per t-MBS in each positive control.
On another day, to confirm the leukocyte ROCK activity, the forearm vascular responses to intra-arterial infusion of fasudil (Asahi Chemical Industry Co., Fukushima, Japan) (3, 10, and 30 μg/min), a specific ROCK inhibitor, were evaluated in 34 of the 242 subjects. Forearm blood flow was measured with a mercury-filled Silastic strain-gauge plethysmography (EC-5R; D.E. Hokanson, Inc. Bellevue, Washington, USA), as described previously.17
Measurement of flow-mediated vasodilation
All studies were performed in the morning, after overnight fasting, in a quiet, dark, air-conditioned room (constant temperature of 22-25°C). The subjects remained supine throughout the study. The vascular response to reactive hyperemia in the brachial artery was assessed for ultrasound assessment of endothelium-dependent flow-mediated vasodilation. A high-resolution linear artery transducer was coupled to computer-assisted analysis software (UNEXEF18G, UNEX Co, Nagoya, Japan) that used an automated edge detection system for measurement of brachial artery diameter. A blood pressure cuff was placed around the forearm. The brachial artery was scanned longitudinally 5-10 cm above the elbow. When the clearest B-mode image of the anterior and posterior intimal interfaces between the lumen and vessel wall was obtained, the transducer was held at the same point throughout the scan by a special probe holder (UNEX Co) to ensure consistency of the image. Depth and gain setting were set to optimize the images of the arterial lumen wall interface. When the tracking gate was placed on the intima, the artery diameter was automatically tracked and the waveform of diameter changes over the cardiac cycle was displayed in real time using the flow-mediated vasodilation mode of the tracking system. This allowed the ultrasound images to be optimized at the start of the scan and the transducer position to be adjusted immediately for optimal tracking performance throughout the scan. Pulsed Doppler flow was assessed at baseline and during peak hyperemic flow, which was confirmed to occur within 15 seconds after cuff deflation. Blood flow velocity was calculated from the color Doppler data and was displayed as a waveform in real time. The baseline longitudinal image of the artery was acquired for 30 seconds and then the blood pressure cuff was inflated to 50 mm Hg above systolic pressure for 5 minutes. The longitudinal image of the artery was recorded continuously until 5 minutes after cuff deflation. Pulsed Doppler velocity signals were obtained for 20 seconds at baseline and for 10 seconds immediately after cuff deflation. Changes in brachial artery diameter were immediately expressed as percent change relative to the vessel diameter before cuff inflation. Flow-mediated vasodilation was automatically calculated as the percent change in peak vessel diameter from the baseline value. %flow-mediated vasodilation (peak diameter - baseline diameter/baseline diameter) was used for analysis. Blood flow volume was calculated by multiplying the Doppler flow velocity (corrected for the angle) by heart rate and vessel cross-sectional area (−r2). Reactive hyperemia was calculated as the maximum percentage increase in flow after cuff deflation compared with baseline flow.
The response to nitroglycerine was used for assessment of endothelium-independent vasodilation. After acquiring baseline rest image for 30 seconds, a sublingual tablet (nitroglycerine 75 μg) was given, and image of the artery was recorded continuously for 5 minutes. Nitroglycerine-induced vasodilation was automatically calculated as a percent change in peak vessel diameter from the baseline value. %nitroglycerine (peak diameter - baseline diameter/baseline diameter) was used for analysis.
Statistical Analysis
Results are presented as mean ± SD. All reported p values were 2-sided, and a p value of <0.05 was considered statistically significant. Relations between variables were determined by Spearman correlation coefficients analysis. Multivariate regression analyses were performed to identify factors associated with ROCK activity or flow-mediated vasodilation in risk factors and laboratory data. The data were processed using the software package Stata version 9 (Stata Co., College Station, Texas, USA).
Results
Baseline clinical characteristics
The baseline clinical characteristics of the 242 subjects are shown in Table 1. Twenty-four (9.9%) of the 242 subjects had hypertension, 17 (7.1%) had diabetes mellitus and 42 (17.4%) had dyslipidemia.
Table 1.
Clinical Characteristics of Subjects
| Variables | n=242 |
|---|---|
| Age, yr | 43 ± 10 |
| Body mass index, kg/m2 | 23.4 ± 3.0 |
| Systolic blood pressure, mm Hg | 118.0 ± 16.7 |
| Diastolic blood pressure, mm Hg | 72.5 ± 11.5 |
| Heart rate, bpm | 67.9 ± 11.9 |
| Total cholesterol, mmol/L | 5.30 ± 0.86 |
| Triglycerides, mmol/L | 1.32 ± 0.78 |
| High-density lipoprotein cholesterol, mmol/L | 1.52 ± 0.41 |
| Low-density lipoprotein cholesterol, mmol/L | 3.22 ± 0.79 |
| Glucose, mmol/L | 5.5 ± 1.2 |
| Hypertension, n (%) | 28 (12) |
| Dyslipidemia, n (%) | 32 (13) |
| Diabetes mellitus, n (%) | 19 (8) |
| Smokers, n | 81 |
All results are presented as mean±SD.
Leukocyte ROCK activity and forearm blood flow response to fasudil
Intra-arterial infusion of fasudil dose-dependently increased forearm blood flow from 4.2±1.2 to 7.4±1.9, 11.9±4.2 and 18.3±6.7 mL/min/100 mL tissue (P<0.05, P<0.001 and P<0.001, respectively) in 34 of the 242 subjects (Figure 1, A). No significant change was found in arterial blood pressure or heart rate with fasudil infusion. There was a significant relationship between leukocyte ROCK activity (p-MBS/t-MBS) and maximal forearm blood flow response to fasudil (r=0.68, P<0.001) (Figure 1, B).
Figure 1.
A, Forearm blood flow (FBF) response to fasudil. *P<0.05, †P<0.001 vs. baseline. B, Relationship between Rho-associated kinase (ROCK) activity in peripheral blood leukocytes and ROCK activity assessed by FBF response to fasudil.
Flow-mediated vasodilation, reactive hyperemic blood flow and vascular response to nitroglycerine
Figure 2 shows representative data on change in reactive hyperemic blood flow (%) and change in brachial artery diameter (%) after cuff deflation. Flow-mediated vasodilation, reactive hyperemic blood flow and nitroglycerine-induced vasodilatation in the subjects are summarized in Table 2.
Figure 2.
Representative data on change in reactive hyperemic blood flow (%) and change in brachial artery diameter (%) after cuff deflation.
Table 2.
Vascular Response of the Subjects
| Variables | n=242 |
|---|---|
| Brachial artery diameter, mm | 4.13 ± 0.52 |
| Increased hyperemic blood flow, % | 421 ± 158 |
| Flow-mediated vasodilation, % | 6.9 ± 3.2 |
| Nitroglycerine-induced vasodilation, % | 10.1 ± 3.8 |
All results are presented as mean±SD.
Leukocyte ROCK activity and cardiovascular risk factors
Figure 3 shows the expression levels of p-MBS, t-MBS, and β tubulin in subjects with low (≤1), intermediate (2 to 6) and high (≥7) Framingham risk factor scores divided into three tertiles. Leukocyte ROCK activity increased with increase in the Framingham risk factor score. The expression levels of t-MBS and β tubulin were similar in subjects regardless Framingham risk factor score.
Figure 3.
Western blot analysis for phospho myosin-binding subunit (p-MBS), total myosin-binding subunit (t-MBS), and β tublin in peripheral blood leukocytes of subjects with low, intermediate, and high Framingham risk scores.
There was a significant positive correlation between leukocyte ROCK activity and Framingham risk factor score (r=0.31, P<0.001) (Figure 4, A). Univariate regression analysis revealed that leukocyte ROCK activity significantly correlated with body mass index (r=0.29, P=0.003), systolic blood pressure (r=0.25, P=0.01), low-density lipoprotein cholesterol level (r=0.21, P=0.04), and flow-mediated vasodilation (r=−0.41, P<0.001) as well as Framingham risk factor score (Table 3, Figure 4, B). Other parameters did not correlate with leukocyte ROCK activity. After adjustments of cardiovascular risk factors that are not included in the components of Framingham risk factor score, Framingham risk factor score remained a statistically significant predictor of leukocyte ROCK activity (β=−0.45, P<0.001). Multivariate analysis revealed that flow-mediated vasodilation was an independent predictor of leukocyte ROCK activity (Table 3).
Figure 4.
A, Relationship between Rho-associated kinase (ROCK) activity in peripheral blood leukocytes and Framingham risk score. B, Relationship between Rho-associated kinase (ROCK) activity in peripheral blood leukocytes and flow-mediated vasodilation. C, Relationship between Rho-associated kinase (ROCK) activity in peripheral blood leukocytes and nitroglycerine-induced vasodilation.
Table 3.
Univariate Analysis of the Relationship between ROCK Activity and Variables
| r | p value | |
|---|---|---|
| Age, years | 0.12 | 0.24 |
| Body mass index, kg/m2 | 0.29 | 0.003 |
| Systolic blood pressure, mm Hg | 0.25 | 0.01 |
| Diastolic blood pressure, mm Hg | 0.17 | 0.11 |
| Heart rate, bpm | 0.11 | 0.27 |
| Total cholesterol, mmol/L | 0.15 | 0.22 |
| Triglycerides, mmol/L | 0.16 | 0.14 |
| High-density lipoprotein cholesterol, mmol/L | −0.06 | 0.55 |
| Low-density lipoprotein cholesterol, mmol/L | 0.21 | 0.04 |
| Glucose, mmol/L | 0.05 | 0.67 |
| Framingham risk score | 0.31 | <0.001 |
| Flow-mediated vasodilation, % | −0.41 | <0.001 |
| Multivariate Analysis of the Relationship between ROCK Activity and Variables | |||
|---|---|---|---|
| β | t value | p value | |
| Age, years | −0.24 | −0.96 | 0.21 |
| Body mass index, kg/m2 | 0.35 | 1.86 | 0.07 |
| Systolic blood pressure, mm Hg | −0.33 | −1.58 | 0.09 |
| Diastolic blood pressure, mm Hg | −0.17 | −0.98 | 0.18 |
| Heart rate (bpm) | 0.02 | 0.21 | 0.84 |
| Total cholesterol, mmol/L | −0.15 | −0.76 | 0.45 |
| High-density lipoprotein cholesterol, mmol/l | 0.04 | 0.23 | 0.81 |
| Low-density lipoprotein cholesterol, mmol/l | −0.31 | −1.29 | 0.20 |
| Triglycerides, mmol/L | 0.19 | 0.92 | 0.37 |
| Glucose (mmol/l) | −0.21 | −1.55 | 0.13 |
| Flow-mediated vasodilation, % | 0.68 | 2.28 | 0.03 |
ROCK indicates Rho kinase activity.
Endothelial function and cardiovascular risk factors
In addition, univariate regression analysis revealed that flow-mediated vasodilation significantly correlated with age (r=−0.23, P=0.02), body mass index (r=−0.19, P=0.05), systolic blood pressure (r=−0.22, P=0.03), total cholesterol level (r=−0.21, P=0.04), low-density lipoprotein cholesterol level (r=−0.22, P=0.03), glucose level (r=−0.20, P=0.04), Framingham risk factor score (r=−0.37, P<0.001), and leukocyte ROCK activity (−0.41, P<0.001) (Table 4). Multivariate analysis revealed that age, systolic blood pressure and ROCK activity were independent predictors of flow-mediated vasodilation (Table 4).
Table 4.
Univariate Analysis of the Relationship between FMD Activity and Variables
| r | p value | |
|---|---|---|
| Age, years | −0.23 | 0.02 |
| Body mass index, kg/m2 | −0.19 | 0.05 |
| Systolic blood pressure, mm Hg | −0.22 | 0.03 |
| Diastolic blood pressure, mm Hg | −0.16 | 0.08 |
| Heart rate, bpm | 0.12 | 0.25 |
| Total cholesterol, mmol/L | −0.21 | 0.04 |
| Triglycerides, mmol/L | −0.16 | 0.09 |
| High-density lipoprotein cholesterol, mmol/L | 0.11 | 0.33 |
| Low-density lipoprotein cholesterol, mmol/L | −0.22 | 0.03 |
| Glucose, mmol/L | −0.20 | 0.04 |
| Framingham risk score | −0.37 | <0.001 |
| Leukocyte Rho kinase actvity | −0.41 | <0.001 |
| Multivariate Analysis of the Relationship between FMD Activity and Variables | |||
|---|---|---|---|
| β | t value | p value | |
| Age, years | 0.49 | 2.39 | 0.02 |
| Body mass index, kg/m2 | −0.17 | −1.09 | 0.21 |
| Systolic blood pressure, mm Hg | 0.41 | 2.22 | 0.04 |
| Diastolic blood pressure, mm Hg | −0.26 | −1.27 | 0.11 |
| Heart rate (bpm) | 0.01 | 0.16 | 0.92 |
| Total cholesterol, mmol/L | −0.18 | −1.12 | 0.20 |
| Triglycerides, mmol/L | 0.09 | 0.42 | 0.77 |
| High-density lipoprotein cholesterol, mmol/l | 0.15 | 0.83 | 0.41 |
| Low-density lipoprotein cholesterol, mmol/l | −0.31 | −1.34 | 0.09 |
| Glucose (mmol/l) | −0.33 | −1.42 | 0.07 |
| Leukocyte Rho kinase activity | 0.59 | 2.58 | 0.004 |
FMD indicates flow-mediated vasodilation.
Discussion
In the present study, we demonstrated that cumulative cardiovascular risk factors increase leukocyte ROCK activity and that flow-mediated vasodilation is an independent predictor of leukocyte ROCK activity in healthy men and those with cardiovascular risk factors but without established cardiovascular or cerebrovascular diseases.
It is well known that cumulative cardiovascular risk contributes to onset of cardiovascular and cerebraovascular outcomes. Hypertension, dyslipidemia, diabetes, aging, smoking, obesity, and menopause are contributing risk factors in cardiovascular and cerebrovascular diseases. These diseases and classical cardiovascular risk factors are associated with endothelial dysfunction.13-21 Level of ROCK activity as well as endothelial function may be a surrogate biologic marker for atherosclerosis and cumulative cardiovascular risk. Increased ROCK activity and endothelial dysfunction may simultaneously or independently affect the maintenance and progression of cardiovascular disease. Indeed, ROCK is known to be activated by several factors such as angiotensin II, thrombin, and a high glucose condition in endothelial cells and/or smooth muscle cells and subsequently to modulate cell contraction, proliferation, apoptosis and gene expression via several signaling pathway including nuclear factor κ B.30-32 In the present study, we revealed that leukocyte ROCK activity is increased in subjects with cardiovascular risk factors, whereas its precise mechanism still remains to be clarified due to the relatively common limitation of clinical studies.
Interestingly, in the present study, we found a significant relationship between leukocyte ROCK activity and flow-mediated vasodilation in relatively healthy subjects. As Sauzeau et al.33 have shown in vascular smooth muscle cells, nitric oxide may also inhibit RhoA translocation from the cytosol to membrane in leukocytes. In addition, previous studies have shown that activation of the RhoA/ROCK pathway impairs nitric oxide bioavailability through downregulation of endothelial nitric oxide synthase mRNA stability, inhibition of endothelial nitric oxide synthase phosphorylation at Ser 1177 via the Akt/PI3K pathway, and enhancement of endothelial nitric oxide synthase phosphorylation at Thr 495.25,26,34,35 Taken together, the results indicate that ROCK activity in leukocytes may reflect ROCK activity in vascular endothelial cells and endothelial function, suggesting an interaction between ROCK activity and endogenous nitric oxide.
ROCK inhibitors such as fasudil and Y-27632 have been used in previous studies to assess ROCK activity in vitro and in vivo.1-5 In a clinical setting, ROCK activity assessed using intra-arterial or intravenous infusion of fasudil was enhanced in patients with angina pectoris, vasospastic angina, pulmonary hypertension, heart failure, and stroke.6-10 ROCK could regulate cell conditions via phosphorylation of downstream target molecules of ROCK such as MBS, adducin, and ezrin/radixin/moesin. Among them, recent studies using a specific antibody to p-MBS on myosin light chain phosphatase, which is a downstream target of ROCK, have clearly demonstrated that ROCK activity (p-MBS/t-MBS) in peripheral leukocytes is enhanced in patients with metabolic syndrome, patients with coronary artery disease, and smokers.36-38 It is clinically important to assess the level of ROCK activity. Both p-MBS/t-MBS in peripheral blood leukocytes and vascular response to the ROCK inhibitor fasudil may be useful for assessing ROCK activity in humans.
We selected measurement of leukocyte p-MBS/t-MBS as a noninvasive method for assessing ROCK activity, since peripheral leukocytes are able to be simply obtained. Measurement of p-MBS/t-MBS in endothelial cells or vascular smooth muscle cells from humans would enable more specific conclusions concerning the role of ROCK activity in cardiovascular diseases to be drawn. Unfortunately, we cannot easily obtain samples of endothelial cells or vascular smooth muscle cells by biopsies under a healthy condition. Several lines of evidence have shown that measurement of leukocyte p-MBS/t-MBS as well as vascular response to the ROCK inhibitor fasudil is useful for assessing ROCK activity.36-38 In the present study, we confirmed a significant correlation between leukocyte p-MBS/t-MBS and vascular response to fasudil. Therefore, we expect that measurement of leukocyte p-MBS/t-MBS will become an index of vascular ROCK activity. Indeed, leukocyte ROCK activity was found to be related to Framingham risk score, which is a risk calculator and an index of cumulative cardiovascular risk commonly used for assessing heart attack or death from heart disease within 10 years. Nevertheless, further studies and technical breakthroughs to evaluate leukocyte ROCK activity are needed to overcome the current problem of measurement of leukocyte ROCK activity being limited to only a few laboratories due to the technical complexity.
In conclusion, Framingham risk score correlated with ROCK activity in peripheral blood leukocytes of healthy men and those with cardiovascular risk factors but without established cardiovascular or cerebrovascular diseases. Cumulative cardiovascular risk factors may enhance ROCK activity, leading to the progression of cardiovascular diseases. Further studies are needed to determine not only whether results of leukocyte ROCK activity measurements in women, who usually have a lower rate of cardiovascular outcomes, are similar to those in men but also whether long-term inhibition of ROCK activity prevents cardiovascular mortality and morbidity.
Acknowledgments
We thank Megumi Wakisaka, Keiko Umeda and Satoko Michiyama for their excellent secretarial assistance.
This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (1859081500 and 21590898) and the National Institutes of Health (HL052233).
Footnotes
Conflict of Interest/Disclosures Dr. James K Liao is a consultant for Asahi-Kasei Pharmaceutical, Inc.
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