The Subendothelial Extracellular Matrix Modulates JNK Activation by Flow

Abstract

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...

Endothelial fluid shear stress sensing in vascular health and disease

“…However, tissue remodeling also involves inflamma- markers such as ICAM-1 or VCAM-1 (66). Mechanistic in vitro studies have shown that fibronectin controls activation of multiple inflammatory mediators in response to disturbed flow (66,67) as well as oxidized LDL, another important inflammatory mediator in atherosclerosis (68). Furthermore, fibronectin suppresses activation of eNOS and production of NO in response to flow (69), thereby inducing endothelial dysfunction.…”

Molecular Biology of Atherosclerosis

“…These include PKC (which has multiple targets), (GCK; or MAP4K2), and PAK1/2 (p21 activated kinase 1 and 2). Flow-induced PAK activation in endothelial cells apparently activates JNK through the MAP2K MKK4 (285,692,1353). JNK induces bone morphogenic protein 2 (BMP2), BMP4, and Toll-like receptor 4 (TLR4) and has numerous other proinflammatory and pro-apoptotic effects in endothelial cells (285) yet only JNK2 appeared to be atherogenic (see TABLE 1).…”

“…Integrins ␣ 5 ␤ 1 and ␣ V ␤ 3 generate proinflammatory outside-in signaling when bound to fibronectin, vitronectin, or fibrinogen. Without these proteins in the ECM, integrin activation does not lead to inflammatory signaling (692,1352). Normal ECM consists primarily of collagen IV and laminin.…”

“…Deletion of the ␣ 2 integrin subunit did not affect atherosclerosis in ApoE KO mice (658), possibly due to overwhelming signaling through ␣ 5 ␤ 1 and ␣ V ␤ 3 integrins as fibronectin accumulated in atherosclerosis-prone areas with age. Conversely, stimulation of the ␣ 5 ␤ 1 and ␣ V ␤ 3 integrins by onset of flow over endothelial cells grown on fibronectin resulted in activation of PKC␣, NF-B, p38, and JNK which inhibited the anti-inflammatory signaling from integrin ␣ 2 ␤ 1 (568,692,1352,1353,1878). Resulting JNK expression primes endothelial cells for apoptosis in atherosclerosis-prone areas (285).…”

Regulation of Adhesion Mechanosignalling by Fibronectin Matrix Structure

“…[51] It has been shown that atheroprone shear stress profiles promote deposition and assembly of fibronectin into the extracellular matrix by activating both fibronectin expression and integrin activation, creating an inflammatory positive feedback loop in the endothelium. Although current studies have linked fibronectin deposition and integrin binding to inflammatory signaling and structural remodeling that may promote atherosclerosis, it remains unknown whether fibronectin assembly can regulate the signaling pathways involved in structural adaptation to shear stress.…”