Measurement of Rho-associated kinase (ROCK) activity in humans: validity of leukocyte p-MBS/t-MBS in comparison with vascular response to fasudil

Abstract

Background: Rho-associated kinases (ROCKs) have been shown to be involved in the pathogenesis of atherosclerosis. It is clinically important to estimate the degree of ROCK activity in humans. The purpose of this study was to confirm the validity of a leukocyte ROCK parameter as an index of ROCK activity in comparison with vascular response to a ROCK inhibitor.

Methods and results: We evaluated the ratio of phospho myosin-binding subunit (p-MBS) on myosin light-chain phosphatase to total MBS in peripheral leukocytes by Western blot analysis and forearm blood flow (FBF) response to the ROCK inhibitor fasudil using strain-gauge plethysmography in 36 healthy subjects and 39 patients with cardiovascular diseases. Fasudil (3, 10, 30μg/min) was infused intra-arterially for 5min at each dose. Leukocyte p-MBS/total-MBS was higher in cardiovascular diseases than in healthy subjects (0.97±0.37 vs. 0.51±0.14; P=0.002). Fasudil increased FBF from 4.9±1.2 to 14.5±5.7mL/min/100mL tissue (P<0.0001) in patients with cardiovascular diseases, while fasudil did not alter FBF in healthy subjects. There was a significant relationship between leukocyte p-MBS/total-MBS and maximal FBF response to fasudil in all subjects (r=0.72, P<0.0001). There was also a significant correlation between p-MBS/total-MBS and maximal FBF response to fasudil in patients with cardiovascular diseases (r=0.59, P<0.0001). In healthy subjects, there was no significant correlation between the two parameters.

Conclusions: These findings suggest that assessment of leukocyte ROCK activity is minimally invasive and does not require pharmacologic intervention using ROCK inhibitors. Leukocyte p-MBS/total-MBS may be useful for evaluating ROCK activity in a clinical setting.

Figure 1

Rho-associated kinase (ROCK) activity in healthy subjects and patients with cardiovascular diseases. Western blot analysis for phospho-myosin-binding subunit (p-MBS), total-myosin-binding subunit (t-MBS), and β tublin (upper). ROCK activity (p-MBS/t-MBS) in healthy subjects and patients with cardiovascular diseases (bottom).

Figure 2

Forearm blood flow response to fasudil in healthy subjects and patients with cardiovascular diseases. *P<0.05 vs. baseline in the same group. †P<0.05 vs. healthy subjects at the same dose.

Figure 3

Relationship between leukocyte phospho-myosin-binding subunit (p-MBS)/total-myosin-binding subunit (t-MBS) and maximal forearm blood flow (FBF) response to fasudil in healthy subjects (open circle) and patients with cardiovascular diseases (closed circle).