Abstract:

The apparent tendency of intracranial aneurysms to form at apices of cerebral bi-furcations and outer sides of curved vessels, has led to many speculations that high wall shear stress (WSS) damages the arterial wall, starting from the outer layer: the endothelial cells (ECs). However, little is known about the EC response to high WSS (>20 dynes/cm2 ). Most in vitro studies have concentrated on WSS values below 15 dynes/cm2 and the in vivo studies, in fact, report opposite EC behaviors at high flow: cell loss during aneurysm initiation and cell proliferation during flow-induced adaptive outward remodeling. This study aims at elucidating the EC growth response to elevated WSS and determining if nitric oxide (NO) and stretch activated cation channels (SACs) are involved. For this purpose, a tapered chamber was designed, where a confluent EC layer was subjected to a wide range of WSS values, from 15 to 100 dynes/cm 2 in a single experiment. The flow experiments ran with steady-state, laminar flow for 24 or 48 hours. Cells oriented to the direction of the flow with a time course that varied with WSS. At 48 hours all cells were aligned with the flow. Cell proliferation was examined using bromodeoxyuridine (BrdU) incorporation. The percentage of proliferating ECs rose linearly from 15 to 50 dynes/cm2 to more than 6-fold at 50-100 dynes/cm 2 compared to the accepted physiological baseline of 15-20 dynes/cm 2 . This result demonstrates that high WSS stimulates EC proliferation. Furthermore, immunostaining revealed increased endothelial nitric oxide synthase (eNOS) production with increasing WSS. Inhibition of NOS with L-NAME drastically reduced the WSS-stimulated proliferation, indicating a critical role of NO production in the stimulation of EC proliferation by high WSS. Moreover, the proper function of SACs proved to be necessary for the eNOS upregulation at high WSS since streptomycin, a SACs blocker, significantly inhibited the increase in eNOS expression. This study is the first to our knowledge that investigates the in vitro molecular EC response to such a wide range of high WSSs. The results show that high WSSs do not damage the ECs but rather stimulate their proliferation through SACs and eNOS upregulation.