The
small G protein RhoA and its downstream effector
Rho-kinase/ROCK2 play an important role in regulation of various vasculature cellular functions.
Nitric oxide (NO) produced by endothelial
NO synthase (eNOS) is an important mediator of vascular homeostasis and cerebral blood flow. Using the human endothelial cell line HUVEC, the present study investigated the role of RhoA and
Rho-kinase in endothelial eNOS
protein expression under hypoxic conditions as an in vitro model of
ischemia.
RhoA protein levels in HUVEC were low under normoxic conditions, but were significantly increased after 5h of
hypoxia. Endothelial
Rho-kinase expression was not detected until after 3h of
hypoxia; such expression remained significantly increased after 5h. On the other hand, endothelial eNOS expression was similar after 3h of
hypoxia, but was significantly decreased after 5h. The
hypoxia-induced decrease in eNOS expression was significantly enhanced by expression of the constitutively active form of RhoA and significantly inhibited by suppression of RhoA expression by
small interfering RNA. The
hypoxia-induced decrease in eNOS expression was significantly inhibited when endogenous
Rho-kinase activation was inhibited by Rho-binding domain expression. Furthermore, the
hypoxia-induced decrease in eNOS expression was significantly enhanced by expression of the constitutively active form of
Rho-kinase. Since expression and activation of RhoA and
Rho-kinase inhibit eNOS expression in endothelial cells, attempts to down-regulate RhoA and
Rho-kinase by multiple drugs, such as
statins or
Rho-kinase inhibitors, might provide endothelial and cardiovascular benefits through upregulation of eNOS.