Genomic analysis of the immediate/early response to shear stress in human coronary artery endothelial cells

The involvement of shear stress in the pathogenesis of vascular disease has motivated efforts to define the endothelial cell response to applied shear stress in vitro. A central question has been the mechanisms by which endothelial cells perceive and respond to changes in fluid flow. We have utilized cDNA microarrays to characterize the immediate/early genomic response to applied laminar shear stress (LSS) in primary cultures of human coronary artery endothelial cells (HCAECs). Cells were exposed, in a parallel plate flow chamber, to 0, 15 or 45 dynes/cm LSS for 1 hour and gene expression profiles determined using human GEM1 cDNA microarrays. We find that a high proportion of LSS-responsive genes are transcription factors and these are related by their involvement in growth arrest. These likely play a central role in the re-programming of endothelial homeostasis following the switch from a static to a shear stressed environment. LSS-responsive genes were also found to encode factors involved in vasoreactivity, signal transduction, anti-oxidants, cell cycle-associated genes and markers of cytoskeletal function and dynamics. (PG-00016-2002.R1) 4 Introduction Physical forces are emerging as major determinants of physiological state and there have been extensive studies describing gene expression changes in response to defined mechanical stress. One area of particular interest is the response of vascular endothelial cells to applied fluid flow. Shear stress is known to elicit modulation in the expression levels of a number of genes involved in vascular reactivity. Based on in vitro analyses, under high fluid shear stresses (>15 dynes/ cm) vascular endothelial cells enter a quiescent, antiproliferative, antioxidant and antithrombotic state which is reflected by the down-modulation of a number of atherogenic factors. For example, high fluid shear stress results in the down-regulation of vascular cell adhesion molecule (VCAM-1), up-regulation of antioxidant genes (Mn-SOD and Cu/ZnSOD), down-regulation of vasoconstrictive factors (ET-1) and up-regulation of vasodilatory factors (eNOS). In contrast, endothelial cells exposed to low or oscillatory fluid shear stress are thought to enter in a pro-coagulant and pro-thrombotic state. Specifically, such conditions have been shown to cause the up-regulation of ET-1, endothelin converting enzyme (ECE), angiotensin converting enzyme (ACE), platelet derived growth factors (PDGF) A and B. Recent developments in genomics have resulted in the availability of techniques for simultaneous parallel expression analysis of multiple genes at the level of transcription. Three recent reports have utilized filter-based microarrays and radioisotope-labeled cDNA to characterize the genomic response of vascular endothelial cells to long-term shear stress exposure. Here we report the use of glass-based microarrays and fluorescently labeled cDNA to characterize the immediate/early transcriptional response of human coronary artery endothelial cells (HCAECs) to different levels of laminar shear stress (LSS) in vitro. This (PG-00016-2002.R1) 5 reveals a rapid transcriptional response after 1 hour of exposure involving modulation of genes representing a number of distinct functional classes. In addition to simultaneously confirming that the antiproliferative, antioxidant, and vasodilatory response to shear stress is realized at the level of transcription within one hour of the onset of flow, we identify numerous transcription factors (TFs) that are likely to be the primary targets of shear stress-responsive signal transduction cascades. Methods Cell culture and exposure to LSS Primary cultures of human coronary artery endothelial cells were purchased from BioWhittaker. Cells were obtained at passage 3 and cultured in EGM2MV medium (BioWhittaker). Cells at passage 5 were seeded at a density of approximately 5 x 10 cells/cm on glass microscope slides and cultured at 37°C in humidified 5% CO2 95% air. Confluent monolayers of cells were then placed in a parallel plate flow chamber under aseptic conditions and perfused in EGM2MV at 37°C in humidified 5% CO2 95% air for 1 hour. Control cells not exposed to LSS were also cultured in EGM2MV for an identical length of time as LSS treated