Immunohistochemical analysis of target proteins of Rho-kinase in a mouse model of accelerated atherosclerosis.
BACKGROUND
The pleiotropic antiatherosclerotic effects of statins are believed to be associated with the inhibition of Rho-kinase. However, a systematic analysis of Rho-kinase activation in atherosclerotic lesions is missing.


OBJECTIVE
To analyze the distribution and phosphorylation of target proteins of Rho-kinase, such as myosin light chain (MLC) and ezrin-radixin-moesin (ERM) proteins, in the apolipoprotein E (ApoE) knockout model of accelerated atherosclerosis, as well as the effects of treatment with the Rho-kinase inhibitor Y-27632.


METHODS
Western diet-fed ApoE-deficient mice underwent carotid ligation and were sacrificed 14 days after surgery. One group of ligated mice was treated with the Rho-kinase inhibitor Y-27632. Nonligated C57Bl6/J mice on normal chow and ApoE-deficient mice on Western diet were used as controls. Lesion structure and size were analyzed using Masson-elastic stained cross-sections. The distribution and phosphorylation of Rho-kinase target proteins were studied immunohistochemically.


RESULTS
Two weeks after surgery, atherosclerotic plaque-like lesions developed in ligated carotids. Lesion development was inhibited by Y-27632. ERM was expressed ubiquitously, but in the intact arteries, it was phosphorylated exclusively in the endothelium and periadventitial adipocytes. In the atherosclerotic lesions, foamy macrophages also exhibited a strong phospho-ERM signal. Y-27632 inhibited ERM phosphorylation in the plaques. MLC and phospho-MLC were associated with smooth muscle cells and did not respond to the Y-27632 treatment.


CONCLUSIONS
A cell type-selective distribution and phosphorylation of target proteins of Rho-kinase were demonstrated in the carotid artery of the normal mouse model, as well as in the ApoE-knockout model of accelerated atherosclerosis. Various downstream targets of the same enzyme may be differentially involved in specific pathological processes in a cell type-specific manner.
Rekhter
Mark
M
Lilly Research Laboratories, Indianapolis, Indiana, USA.
Chandrasekhar
Kaarthik
K
Gifford-Moore
Donetta
D
Huang
Xiao-di
XD
Rutherford
Pamela
P
Hanson
Jeffrey
J
Kauffman
Raymond
R