Role of Shear Stress in Endothelial Cell Morphology and Expression of Cyclooxygenase Isoforms

Abstract

Objective-The goal of this study was to examine the effect of chronic heterogeneous shear stress, applied using an orbital shaker, on endothelial cell morphology and the expression of cyclooxygenases 1 and 2. Methods and Results-Porcine aortic endothelial cells were plated on fibronectin-coated Transwell plates. Cells were cultured for up to 7 days either under static conditions or on an orbital shaker that generated a wave of medium inducing shear stress over the cells. Cells were fixed and stained for the endothelial surface marker CD31 or cyclooxygenases 1 and 2. En face confocal microscopy and scanning ion conductance microscopy were used to show that endothelial cells were randomly oriented at the center of the well, aligned with shear stress nearer the periphery, and expressed cyclooxygenase-1 under all conditions. Lipopolysaccharide induced cyclooxygenase-2 and the production of 6-ketoprostaglandin F 1␣ in all cells. Key Words: eicosanoids Ⅲ endothelium Ⅲ prostacyclin Ⅲ cyclooxygenase Ⅲ shear stress E ndothelial cells line the luminal surface of blood vessels and are continuously exposed to hemodynamic shear stress. The level of shear stress that cells experience varies from region to region within the vasculature. In areas of high laminar shear stress, endothelial cells are elongated, aligned, and protected from inflammation. In areas of low, oscillatory shear stress, endothelial cells are randomly orientated and susceptible to inflammation. Areas of low shear stress are thought to be atheroprone, whereas areas of high shear stress are thought to be atheroprotected. [1][2][3] Cultured endothelial cells are routinely studied under static conditions, where they appear nonaligned, with a cobblestone morphology. 4,5 It is increasingly recognized that endothelial cells grown under static conditions may not be representative of endothelial cells in the body. 6,7 In addition, evidence suggests that endothelial endocrine function and expression of key enzymes, including cyclooxygenase (COX), is also regulated by shear stress. 8 COX is present in 2 isoforms: COX-1 and COX-2. Generally, COX-1 is expressed constitutively, whereas COX-2 is induced at sites of inflammation. 9 In endothelial cells, COX-1 activity results predominantly in the production of the antithrombotic hormone prostacyclin. 10 COX-1 and COX-2 are the targets for nonsteroidal antiinflammatory drugs (NSAIDs). They have attracted much media attention since the association of COX-2-selective NSAIDs with adverse cardiovascular events, 11,12 although the mechanism behind this association remains unclear. One leading hypothesis is that COX-2-selective drugs reduce the production of the cardioprotective hormone prostacyclin, 13,14 which, in susceptible individuals, increases the risk of arterial thrombosis. Prostacyclin is formed mainly by endothelial cells, which express high levels of COX. It has previously been shown that COX-1 predominates over COX-2 in endothelial cells cultured under static conditions, 15-17 which raises the question of how COX-2-s...

Cyclooxygenase-1, not cyclooxygenase-2, is responsible for physiological production of prostacyclin in the cardiovascular system

“…This conclusion regarding the regulation of expression of COX-2 by shear, however, is based upon studies using short periods of shear stress, which may be perceived by cells as an inflammatory insult that resolves with time. Previous work from our group has shown that chronic exposure to shear stress (up to 7 d) is not associated with increases in COX-2 expression in porcine aortic endothelial cells (25). Here we confirm our earlier observations, showing that COX-1, but not COX-2 immunoreactivity was abundant in human aortic endothelial cells cultured under static conditions (Fig.…”

Disturbed Flow Promotes Endothelial Senescence via a p53-Dependent Pathway

“…Moreover, cells in the central region were not aligned, whereas cells in the periphery were elongated and aligned in the direction of the flow ( Figure 3C) as described previously. 40,41 We conclude that the orbital flow system generates spatially distinct flow fields and that cells in the center display morphological and transcriptional features that are characteristic of the response to disturbed flow. We used the orbital shaker platform to determine whether the application of disturbed or undisturbed flow for Figure 3C and Figure IIA in the online-only Data Supplement).…”

“…To test this, we used an orbital shaking platform which generates reproducible spatially separated biaxial (disturbed) and uniaxial pulsatile (undisturbed) flow patterns. [39][40][41] CFD (computational fluid dynamics) analysis demonstrated that orbital shaking generated wall shear stress at the center of the well that had a relatively constant low magnitude but varied rapidly in direction ( Figure 3A). Conversely, the periphery was associated with temporal fluctuations in the magnitude of wall shear stress, a relatively high maximum wall shear stress and relatively uniform direction ( Figure 3A).…”

Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

“…This has been seen in areas such as inducing changes in cell shape and orientation, secretion and organization, proliferation and differentiation, intracellular signaling, cytoskeleton protein production and gene expression, vessel maturation and structure, and barrier functions [37][38][39][40][41][42][43][44][45][46][47] . The current in vitro methods for forming endothelial tubes generally rely on endothelial cells' (ECs) self-organization with or without mesenchymal cells in extracellular matrix (ECM) (type I collagen, fibrin, or Matrigel) [48][49][50][51][52][53][54] .…”