Rho kinase (ROCK) has been implicated in the regulation of vascular tone, endothelial dysfunction,
inflammation and remodeling. Endothelial progenitor cells (
EPC) have been proven to have the efficacy of therapeutic neovascularization in
ischemia. However, the scarcity of EPCs limits
cell therapy. Using an in vitro
EPC culture assay,
Y27632 was found to increase the number of adherent EPCs. In this study, we investigated the effect of
fasudil, another ROCK inhibitor being used in the clinic, on
EPC number and examined whether EPCs expanded by
fasudil are functional in vitro and in vivo. In ex vivo cultures of EPCs,
fasudil effectively increased the number of ac-
LDL/UEA-1 positive cells as well as adherent cells, in contrast to
H89, a less selective ROCK inhibitor.
Fasudil also increased
EPC numbers in culture up to 10 µM, in a dose-dependent manner. When EPCs expanded with
fasudil were examined for the migratory activity toward stromal cell-derived factor-1 and
vascular endothelial growth factor, these cells retained functional properties in migration, albeit with some decrease.
Fasudil-cultured EPCs labeled with
PKH26 showed an activity similar to non-treated EPCs for cellular adhesion into an endothelial cell (EC) monolayer and incorporation into capillary-like structures formed by ECs. Finally, when EPCs cultured with
fasudil (106 cells/mouse) were injected into ischemic limbs, these cells showed a blood flow recovery at a level comparable to non-treated control EPCs and increased neovascularization. Therefore, these data suggest that the ROCK inhibitor
fasudil can provide a beneficial effect in the treatment of ischemic diseases by increasing
EPC numbers.