Endothelial Cells under High Wall Shear Stress and Spatial Gradients
Dolan, Jennifer M.
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An intracranial aneurysm (IA) is a pathological bulging or dilation of a cerebral vessel which develop at apices of bifurcations or the outer sides of curved vessels. These locations represent a special hemodynamic environment where flow impingement causes a high frictional force (> 10 Pa) on the vessel wall known as wall shear stress (WSS) and significant flow acceleration or deceleration, creating positive or negative spatial gradient of WSS (WSSG). By mapping hemodynamics onto histology during IA initiation in rabbits it has been demonstrated that aneurysms form where WSS is high and WSSG is positive, but not where WSS is equally high and WSSG is negative. Because WSS and WSSG are felt by endothelial cells (ECs), we examined how ECs respond to high WSS (>10 Pa) and WSSG in vitro and in vivo. DNA microarrays were run on cul-tured bovine aortic ECs exposed to no flow (0 Pa), normal WSS (2 Pa) and high WSS (10 Pa) for 24 hrs in vitro. 10 Pa WSS induced gene expression distinct from both 0 and 2 Pa. Gene ontology and biological pathway analysis revealed that high WSS was anti-coagulant, anti-inflammatory, proliferative and pro-matrix remodeling. Based on the gene expression profiling of cultured ECs, we predicted that expansive remodeling in response to high flow involves endothelial cell production of the matrix-degrading enzymes expressed in vitro. To test this, the ex-pressions of ADAMTS1 and uPA proteins, which are both involved in matrix processing, were assessed by immunohistochemistry in rabbit basilar arteries experiencing increased flow after bilaterial carotid artery ligation. Both of these proteins were significantly increased when WSS was elevated compared to sham operated animals. We next considered the effects of WSSG using a flow chamber with converging and diverging channels to vary WSS between 3.5 and 28.4 Pa, creating positive and negative WSSG. High WSS alone increased EC proliferation and apoptosis and inhibited EC alignment to flow. These responses were either enhanced or diminished by WSSG: Positive WSSG (+980 Pa/m) stimulated proliferation (assessed by BrdU incorporation) and apoptosis (determined using a TUNEL assay) and inhibited alignment, whereas negative WSSG (-1120 Pa/m) suppressed proliferation and apoptosis, and promoted alignment. Having established that ECs sense positive vs. negative WSSG, we ran microarrays to identify genes modulated specifically by WSSG. Analysis of genes expressed by ECs exposed to positive WSSG or negative WSSG for 24 hours revealed that positive WSSG favored proliferation, apoptosis, and extracellular matrix processing and decreased expression of pro-inflammatory genes. To begin learning how these pathways might function in IA initiation, we examined EC proliferation and expression of the matrix metalloproteinase, ADAMTS1, under high WSS and WSSG created at the basilar terminus of rabbits after carotid ligation. WSSG at the bifurcation was determined by computational fluid dynamic simulations from 3D angiography and then mapped on immunofluorescent staining for ADAMTS1 or Ki-67, a proliferation marker. Both ADAMTS1 and proliferation were significantly higher in ECs under positive WSSG than in adjacent regions of negative WSSG. High levels of proliferation under positive WSSG may indicate increased cell turnover, which could contribute to aneurysm initiation by causing endothelial dysfunction; e.g., creating transient gaps in the endothelium or diverting ECs from the production of signals that maintain quiescence of smooth muscle cells in the media.