Atherosclerosis occurs mainly at regions of vessel curvature, branch points and bifurcations. These regions are distinguished by blood flow patterns with features such as low shear stress, flow separation and reattachment, and flow reversal during the cardiac cycle (so-called oscillatory flow)
]. Application of analogous flow patterns to endothelial cells (ECs) in vitro recapitulates many of the effects seen in vivo at atherosclerosis-prone sites, including inflammatory gene expression, increased permeability, leukocyte recruitment and cell turnover
]. In vivo, these atherosclerosis-prone regions of arteries show mild chronic inflammation without plaque formation even in WT mice or other organisms that do not normally develop atherosclerosis
]. Progression to atherosclerotic plaque requires additional risk factors such as hyperlipidemia or diabetes
]. Taken together, these results suggest that biomechanical activation of the endothelium by disturbed flow is the initiating event that determines the spatial distribution of atherosclerotic plaques, whereas systemic risk factors contribute mainly to the progression of mild inflammation into atherosclerosis
p21-activated kinases (PAKs) comprise a highly conserved family of serine/threonine protein kinases that are important effectors of Rac and Cdc42. PAKs have been implicated in proliferative signaling by growth factor receptor tyrosine kinases, in control of cell polarity and actin cytoskeletal organization in higher eukaryotic cells
]. On the basis of biochemical and structural features, PAKs are classified into Group I, consisting of PAK1-3, and Group II, consisting of PAK4-6
]. Among group I PAKs, PAK1 and -2 are widely expressed, including in endothelial cells, whereas PAK3 is found largely in the brain. PAK substrates include components of the mitogen-activated protein (MAP) kinase pathway, cytoskeletal proteins such as filamin and Op18, regulators of cell survival, and the transcription factor nuclear factor (NF) – κB.
Previous work from our lab showed that in endothelial cells, application of fluid shear stress activates PAK, which subsequently controls junctional integrity and vascular permeability
]. Moreover, PAK is a critical modulator of several inflammatory pathways, including NF-κB and JNK, such that PAK activity is required for flow-dependent activation of these pathways and induction of downstream genes. Furthermore, PAK activation in mouse arteries was observed specifically in atherosclerosis-prone sites and correlated with inflammatory gene expression, including vascular cell adhesion molecule-1 (VCAM-1). PAK inhibitors reduced NF-κB and JNK phosphorylation in ECs in response to fluid shear stress in vitro, and reduced vascular permeability in vivo, indicating its functional significance in these processes.
Remodeling of the subendothelial extracellular matrix (ECM) appears to be an important component of atherosclerotic progression
]. Fibronectin localizes to the subendothelial ECM selectively in atherosclerosis-prone regions in vivo. In vitro, ECs on basement membrane proteins show reduced inflammatory activation in response to flow and other stimuli. By contrast, cells on fibronectin, an ECM protein associated with remodeling, injury and inflammation, respond strongly to the same stimuli. PAK showed enhanced activation by flow, oxidized LDL and MCP-1 in cells on fibronectin compared to basement membrane proteins, and this differential activation of PAK mediates the ECM-specific activation of NF-κB and JNK
]. These effects form part of a positive feedback loop in which inflammatory pathways promote fibronectin gene expression and matrix assembly, which subsequently enhances inflammation. Genetic studies in mice and humans support a causal role for ECM remodeling in atherosclerosis
Previous studies of PAK in these processes were based on the use of PAK inhibitors that are isoform nonspecific and may have other non-specific effects. Therefore, to further define the role of PAK in the biomechanical initiation of the atherosclerotic cascade, we examined inflammatory activation of the endothelium at atherosclerosis-prone sites in PAK-1 knockout mice. These studies suggest that PAK1 plays a significant role in initiation of atherosclerosis.