The Role of Hemodynamics in Modulating Kaiso Expression and Activity in Atherogenesis

Atherosclerosis is an inflammatory disease of the vasculature characterized by the accumulation of lipids beneath the endothelium leading to the formation of plaque. The generation of atherosclerosis has been shown to be a non-random process, with endothelial cells (ECs) in regions of disturbed flow, as dictated by variations in regional vascular geometry, developing an atheroprone phenotype when compared to endothelium in regions of nondisturbed, atheroprotective flow. Consequently, hemodynamics (i.e. shear stress) provide a vital mechanical signal that regulates regional susceptibility to atherosclerosis. While a great deal of research has focused on shear stress regulation of EC phenotype as it pertains to atherogenesis, large gaps remain in the understanding of protein expression and signaling pathways that mediate ECs’ response to local hemodynamic environment. We hypothesized that hemodynamic forces modulate the expression and/or activity of the transcription factor Kaiso in a manner that alters local endothelial susceptibility to atherogenesis.

To investigate the effects of hemodynamics on Kaiso function, we utilized a novel in vitro system to apply physiologically determined shear stresses from a human common carotid artery (atheroprotective) or internal carotid sinus (atheroprone) to primary human endothelial cell (EC) monolayers. Through the evaluation of a luciferase-based reporter, we demonstrated that Kaiso activity is differentially regulated by hemodynamic conditions in a manner that is independent of changes in Kaiso expression or interaction with its known activity modulator p120 catenin. Having established Kaiso’s dependence on hemodynamic environment, we sought to elucidate the functional role of Kaiso in mediating transcription pathways downstream of physiological shear stress exposure. To obtain a broad characterization of Kaiso function in endothelial cells, siRNA was used to knockdown Kaiso expression in ECs exposed to atheroprotective or atheroprone hemodynamics, and changes in the transcriptome were assayed by gene microarrays. The microarrays demonstrated involvement of Kaiso in a variety of relevant signaling pathways and suggested an elevated importance of Kaiso in mediating the downstream effects of atheroprotective hemodynamics. In order to ascertain a more specific understanding of Kaiso function in response to shear stress, potential atherosclerosis-related Kaiso gene targets were identified through a bioinformatic analysis of human promoter sequences. We then investigated the response of these genes to either Kaiso knockdown or overexpression in the context of hemodynamic environment. Our findings reveal a complex and possibly bimodal activity of Kaiso that is dependent on both hemodynamic environment and loss or gain of expression.

While Kaiso demonstrated many varied functions in endothelium, our studies specifically established a novel role for Kaiso as a positive regulator of the highly anti-atherogenic KLF2/KLF4 signaling pathway downstream of exposure to atheroprotective hemodynamics. Kaiso knockdown resulted in a downregulation of eNOS, KLF2, KLF4, MEF2a, NOV, TFPI, and THBD, all of which are known mediators of anti-inflammatory and anti-thrombotic effects. This loss of atheroprotective expression correlated with increased inflammatory NFκB activity and subsequently elevated expression of the leukocyte adhesion molecules VCAM-1 and ICAM-1. To support the physiological significance of Kaiso-mediated atheroprotection, we confirmed that knockdown of Kaiso expression is sufficient to increase monocyte adhesion to EC monolayers. Taken together, these results indicate a potentially critical role for Kaiso in conferring atheroprotection to endothelium in response protective hemodynamic exposure. This finding improves our understanding of hemodynamic modulation of athero-susceptibility and may serve to generate future therapeutic targets for the treatment and prevention of atherosclerosis.