Abstract-Atherosclerosis begins as local inflammation of artery walls at sites of disturbed flow. JNK (c-Jun NH 2 -terminal kinase) is thought to be among the major regulators of flow-dependent inflammatory gene expression in endothelial cells in atherosclerosis. We now show that JNK activation by both onset of laminar flow and long-term oscillatory flow is matrix-specific, with enhanced activation on fibronectin compared to basement membrane protein or collagen. Flow-induced JNK activation on fibronectin requires new integrin ligation and requires both the mitogen-activated protein kinase kinase MKK4 and p21-activated kinase. In vivo, JNK activation at sites of early atherogenesis correlates with the deposition of fibronectin. Inhibiting p21-activated kinase reduces JNK activation in atheroprone regions of the vasculature in vivo. These results identify JNK as a matrix-specific, flow-activated inflammatory event. Together with other studies, these data elucidate a network of matrix-specific pathways that determine inflammatory events in response to fluid shear stress. Key Words: shear stress Ⅲ atherosclerosis Ⅲ JNK A therosclerosis is an inflammatory disease in which endothelial cell (EC) activation leads to leukocyte recruitment into artery walls, followed by formation of plaques containing lipid-laden macrophages and smooth muscle cells. 1 Plaques can occlude vessels and cause ischemia, or rupture to cause stroke or myocardial infarction. Whereas systemic risk factors such as hypertension, smoking, and obesity play important roles in atherogenesis, plaques show a predilection for vessel branch points and regions of high curvature, where flow is low and shows a variety of complex patterns that are grouped together under the term disturbed flow. 2 These areas show increased EC turnover, altered redox regulation, and upregulation of proinflammatory genes that contribute to atherosclerotic progression. 3,4 By contrast, areas of high laminar shear show downregulation of proatherogenic genes and upregulation of atheroprotective genes and are resistant to atherosclerosis. 5 In vitro, acute application of laminar flow to unstimulated cells transiently activates inflammatory events and is often used to investigate EC responses to flow. Interestingly, oscillatory flow activates most of the same events in a sustained manner, recapitulates features of atheroprone regions of arteries in vivo, and has been used to model complex flow profiles found in vivo. Together, these methods have been widely used to study flow signaling associated with atherosclerosis.Previous work in our laboratory showed that integrins are converted to a high affinity state downstream of a cell-cell junction mechanosensory complex consisting of platelet endothelial cell adhesion molecule-1, VE-cadherin, and vascular endothelial growth factor receptor 2. 6 In response to shear stress, this complex stimulates phosphoinositide 3-kinase (PI3K), which then leads to integrin activation. Subsequent binding of newly activated integrins to extracellular matr...

genesis involves activation of NF-B in endothelial cells by fluid shear stress. Because this pathway involves integrins, we investigated the involvement of focal adhesion kinase (FAK). We found that FAK was not required for flow-stimulated translocation of the p65 NF-B subunit to the nucleus but was essential for phosphorylation of p65 on serine 536 and induction of ICAM-1, an NF-B-dependent gene. NF-B activation by TNF-␣ or hydrogen peroxide was FAK independent. Events upstream of NF-B, including integrin activation, Rac activation, reactive oxygen production, and degradation of IB, were FAK independent. FAK therefore regulates NF-B phosphorylation and transcriptional activity in response to flow by a novel mechanism.atherosclerosis; fluid shear stress; integrin signaling; mechanotransduction CURRENT MODELS FOR ATHEROSCLEROSIS suggest that local endothelial dysfunction results in monocyte recruitment and lipid deposition in the vessel intima (29). In addition to systemic risk factors, fluid shear stress from flowing blood plays a crucial role. Atherosclerotic lesions develop preferentially in regions of oscillatory or disturbed flow that have lower mean shear stress, multidirectionality, and flow separation (33).The proinflammatory transcription factor NF-B is thought to be a key determinant of atherogenesis (8). NF-B is activated in atheroprone regions in vivo concomitant with expression of its target genes including ICAM-1, VCAM-1 (20), monocyte chemoattractant protein 1, tissue factor, and PDGF (8). NF-B is a dimer, usually consisting of a p65 and a p50 subunit, though other combinations also exist. Inactive NF-B is sequestered in the cytoplasm by IB proteins (8,20). The IB kinase (IKK) complex phosphorylates IB, leading to its ubiquitination and proteosomal degradation. Free NF-B dimer then translocates into the nucleus and binds to B enhancer sites. Phosphorylation, acetylation, and sumoylation regulates the subsequent recruitment of cofactors and other members of the transcriptional machinery required to drive target gene expression.Integrins have been implicated in activation of NF-B and several other proinflammatory pathways in response to flow (19). Shear stress induces conversion of integrins to a high-affinity state (i.e., integrin activation), which is followed by binding of these high-affinity integrins to the extracellular matrix beneath the cells to form new integrinmatrix contacts (53). The newly ligated integrins trigger downstream signals, including RhoA, Rac, and Cdc42 activation (53-55). These GTPases mediate cell alignment, sterol regulatory element-binding protein (SREBP) cleavage (30), and . Consistent with this model, flow-induced activation of NF-B is integrin and matrix dependent, occurring in cells on fibronectin (FN) or fibrinogen but not on collagen or laminin (38).Focal adhesion kinase (FAK), a nonreceptor tyrosine kinase involved in integrin signaling (43,46), is present in endothelial cells and has been implicated in responses of endothelial cells to fluid shear stress. Complete ...

Key Words: shear stress Ⅲ extracellular matrix Ⅲ protein kinase A Ⅲ p21-activated kinase Ⅲ NF-B A therosclerosis, a chronic inflammatory disease of artery walls, originates as regions of local endothelial cell dysfunction characterized by enhanced permeability, inflammatory gene expression, and turnover. 1 Although classic risk factors for atherosclerosis, including hypercholesterolemia, hyperglycemia, and smoking occur throughout the vasculature, atherosclerotic plaques preferentially form at vessel curvatures, branch points, and bifurcations, where blood flow is of lower magnitude and exhibits complex features including turbulence, oscillations, separation, and reattachment, which we term disturbed flow. 2 Thus, flow patterns critically regulate the local susceptibility to atherosclerosis.In vitro, laminar flow inhibits endothelial activation and turnover, whereas disturbed flow induces inflammatory signaling, enhanced turnover, and other features of atherosclerosis-susceptible regions of arteries. 3 Interestingly, laminar and disturbed flow both initially activate nuclear factor (NF)-B 4 and JNK (c-Jun N-terminal kinase), 5 intercellular adhesion molecule (ICAM)-1 expression, 6 and enhanced permeability. 7 However, in laminar flow, these events decrease at later times as the cells align in the direction of flow, whereas in disturbed flow, these events remain elevated. 3 Thus, the inability of cells to adapt to disturbed flow may mediate the differential cellular responses to these 2 flow patterns.Integrins mediate an important subset of the endothelial response to flow. Shear stress stimulates integrin activation, 8,9 conversion from a low affinity to a high affinity state, which triggers new integrin-matrix binding; integrins thereby regulate both flow-induced endothelial cell alignment and proinflammatory gene expression. 10 Individual integrin heterodimers differ in both their ligand preferences and signaling Original received September 30, 2009; revision received February 22, 2010; accepted February 25, 2010 7 and PAK mediates matrix-specific activation of the proinflammatory transcription factor NF-B, 13,14 inflammatory gene expression, 13 and increased endothelial permeability 7 by flow. PAK is activated in atheroprone regions in vivo, which correlates with areas of fibronectin deposition and inflammatory gene expression. 7 However, the mechanisms mediating matrix-specific PAK activation remain unknown.PAK is activated by the small GTPases Rac and Cdc42, 12 and suppressed by PAK inhibitory proteins (eg, nischarin, hPIP1), 15,16 by dephosphorylation by phosphatases (PP2A, POPX1/2), 17,18 and by phosphorylation by protein kinase (PK)A. 19 We therefore set out to elucidate the mechanism of differential PAK activation on different matrix proteins. Our results identify PKA as the critical mediator of matrixspecific PAK activation and hence proinflammatory signaling through NF-B. MethodsBriefly, bovine aortic endothelial cells (BAECs) and human aortic endothelial cells (HAECs) on glass slides coated with s...

Human immunodeficiency virus dementia (HIV-D) is a nonfocalcentral nervous system manifestation characterized by cognitive, behavioral, and motor abnormalities. The pathophysiology of neuronal damage in HIV-D includes a direct toxic effect of viral proteins on neuronal cells and an indirect effect caused by the release of inflammatory mediators and neurotoxins by activated macrophages/microglia and astrocytes, culminating into neuronal apoptosis. Previous studies have documented that the nucleoside adenosine mediates neuroprotection by activating adenosine A 1 receptor subtype (A 1 AR) linked to suppression of neuronal excitability. In this study, we show that A 1 AR activation protects against HIV-1 Tat-induced toxicity in primary cultures of rat cerebellar granule neurons and in rat pheochromocytoma (PC12) cell. In PC12 cells, HIV-1 Tat increased [Ca 2ϩ ] i levels, release of nitric oxide (NO), and expression of inducible nitric-oxide synthase (iNOS) and A 1 AR. Activation of A 1 AR suppressed Tat-mediated increases in [Ca 2ϩ ] i and NO. Furthermore, A 1 AR agonists inhibited iNOS expression in a nuclear factor-B (NF-B)-dependent manner. It is noteworthy that activation of the A 1 AR or inhibition of NOS protected against Tat-induced apoptosis in PC12 cells and cerebellar granule cells. Moreover, activation of the A 1 AR-inhibited Tat-induced increases in the levels of proapoptotic proteins Bax and caspase-3. Taken together, our results demonstrate that the A 1 AR protects against HIV-1 toxicity by inhibiting NF-B, thereby reducing the expression of iNOS and NO radicals and neuronal apoptosis.