Homeodomain interacting protein kinase 2 activation compromises endothelial cell response to laminar flow: Protective role of p21waf1,cip1,sdi1

Background: In the cardiovascular system, laminar shear stress (SS) is one of the most important source of endothelial protecting signals. Physical and chemical agents, however, including ionising radiations and anticancer drugs, may injure endothelial cells determining an increase in oxidative stress and genotoxic damage. Whether the SS protective function remains intact in the presence of strong oxidants or DNA damage is currently unclear. Methods and Results: To investigate this aspect a series of experiments were performed in which HUVEC were exposed to sub-lethal doses of the radio-mimetic compound Bleomycin (Bleo; 10 μg/ml) which generated free radicals (ROS) without significantly compromising cell survival. Remarkably, the application of a SS of 12 dyne/cm² did not protect endothelial cells but markedly accelerated apoptosis compared to controls kept in static culture and in the presence of Bleo. Experiments with the inducible nitric oxide synthase (iNOS) inhibitor GW274150 significantly reduced the SS-dependent apoptosis indicating that the production of NO was relevant for this effect. At molecular level, the ataxia-telangectasia-mutated (ATM) kinase, the homeodomain-interacting protein kinase-2 (HIPK2) and p53 were found activated along a pro-apoptotic signalling pathway while p21^{waf1,cip1,sdi1} was prevented from its protective action. RNA interference experiments revealed that HIPK2 and p53 were both important for this process, however, only the forced expression p21^{waf1,cip1,sdi1} fully restored the SS-dependent pro-survival function. Conclusions: This study provides the first evidence that, in the presence of genotoxic damage, laminar flow contributes to endothelial toxicity and death and identifies molecular targets potentially relevant in endothelial dysfunction and cardiovascular disease pathogenesis.