αvβ3 Integrins Mediate Flow-Induced NF-κB Activation, Proinflammatory Gene Expression, and Early Atherogenic Inflammation

Abstract

Endothelial cell interactions with transitional matrix proteins, such as fibronectin, occur early during atherogenesis and regulate shear stress-induced endothelial cell activation. Multiple endothelial cell integrins bind transitional matrix proteins, including a5b1, avb3, and avb5. However, the role these integrins play in mediating shear stress-induced endothelial cell activation remains unclear. Therefore, we sought to elucidate which integrin heterodimers mediate shear stress-induced endothelial cell activation and early atherogenesis. We now show that inhibiting avb3 integrins (S247, siRNA), but not a5b1 or avb5, blunts shear stress-induced proinflammatory signaling (NF-kB, p21-activated kinase) and gene expression (ICAM1, VCAM1). Importantly, inhibiting avb3 did not affect cytokine-induced proinflammatory responses or inhibit all shear stress-induced signaling, because Akt, endothelial nitric oxide synthase, and extracellular regulated kinase activation remained intact. Furthermore, inhibiting av integrins (S247), but not a5 (ATN-161), in atherosclerosis-prone apolipoprotein E knockout mice significantly reduced vascular remodeling after acute induction of disturbed flow. S247 treatment similarly reduced early diet-induced atherosclerotic plaque formation associated with both diminished inflammation (expression of vascular cell adhesion molecule 1, plaque macrophage content) and reduced smooth muscle incorporation. Inducible, endothelial cell-specific av integrin deletion similarly blunted inflammation in models of disturbed flow and diet-induced atherogenesis but did not affect smooth muscle incorporation. Our studies identify avb3 as the primary integrin heterodimer mediating shear stress-induced proinflammatory responses and as a key contributor to early atherogenic inflammation. (Am J Pathol 2015 http://dx.doi.org/10.1016/j.ajpath.2015 Although traditional risk factors for atherosclerosis, such as hypercholesterolemia and hyperglycemia, are systemic throughout the circulation, atherosclerotic plaques form at discrete areas of the vasculature where vessel geometry results in altered hemodynamics.1,2 Endothelial cells respond to the frictional force generated by these flow patterns, termed shear stress, and convert them into intracellular biochemical signals that critically modulate endothelial cell function. In straight regions of arteries, shear stress generated by unidirectional, laminar flow promotes nitric oxide production and limits endothelial cell activation, consistent with the absence of atherosclerosis in these areas.1,2 In contrast, shear stress generated by disturbed flow patterns, such as those observed at sites of vessel branch points, bifurcations, and curvatures, results in endothelial cell activation with enhanced proinflammatory gene expression [intercellular adhesion molecule-1 (ICAM1), vascular cell adhesion molecule-1 (VCAM1)] and permeability.

Integrin signaling in atherosclerosis

“…Macrovascular endothelial cells express multiple basement membrane binding integrins (Table I), including α2β1, α3β1, α6β1, and α6β4, and signaling through these integrins is widely associated with a quiescent endothelial cell phenotype [14, 33, 34]. During the early stages of atherosclerosis, provisional matrix proteins, such as the RGD-containing proteins fibronectin, fibrinogen, and thrombospondin-1, show significantly enhanced deposition within the endothelial cell matrix [31, 35, 36].…”

The arterial microenvironment: the where and why of atherosclerosis

“…Activation of disparate signaling responses, through integrin-specific recruitment of certain signaling partners, generates contextual cues that affect cell function and integrate with other microenvironmental stimuli [15]. Arterial endothelial cells interact with collagen IV and laminins through the integrins α2β1, α3β1, α6β1, and α6β4 [16]. SMCs interact with their matrix through the collagen-binding integrins α1β1 and α2β1 and the laminin-binding integrins α3β1 and α7β1, although other non-integrin receptors also mediate this interaction including the collagen-binding discoidin domain receptors (DDRs) and the laminin-binding receptor α-dystroglycan [17].…”

“…Similarly, shear stress promotes the activation of both α5β1 and αvβ3 [84, 85]. However, only αvβ3 expression is required for shear stress-induced NF-кB activation and proinflammatory gene expression [16]. Like NF-кB, shear stress-induced PAK activation requires αvβ3 [16] and the presence of provisional matrix proteins [59, 61], and endothelial PAK activation corresponds to early fibronectin deposition at sites of disturbed flow in vivo [61].…”

“…However, only αvβ3 expression is required for shear stress-induced NF-кB activation and proinflammatory gene expression [16]. Like NF-кB, shear stress-induced PAK activation requires αvβ3 [16] and the presence of provisional matrix proteins [59, 61], and endothelial PAK activation corresponds to early fibronectin deposition at sites of disturbed flow in vivo [61]. In contrast, endothelial cells on basement membrane proteins show enhanced shear stress-induced PKA activation and NO production [47, 86], associated with reduced PKA-mediated eNOS phosphorylation [47].…”

“…Smooth muscle cells express multiple provisional matrix-binding integrins; however, αvβ3 and αvβ5 show particular upregulation in neointimal SMCs [129]. Treatment with inhibitors of αvβ3 but not α5β1 phenocopies the effect of plasma fibronectin deletion, suggesting that fibronectin promotes SMC proliferation through αvβ3 [16, 25]. Consistent with this αvβ3 dominant role, inhibitory antibodies against αvβ3 reduces plaque development in diabetic pigs associated with diminished plaque SMC content [130].…”