A clear relationship between diabetes and cardiovascular disease has been established for decades. Despite this, the mechanisms by which diabetes contributes to plaque formation remain in question. Some of this confusion derives from studies in type 2 diabetics where multiple components of metabolic syndrome show proatherosclerotic effects independent of underlying diabetes. However, the hyperglycemia that defines the diabetic condition independently affects atherogenesis in cell culture systems, animal models, and human patients. Endothelial cell biology plays a central role in atherosclerotic plaque formation regulating vessel permeability, inflammation, and thrombosis. The current paper highlights the mechanisms by which hyperglycemia affects endothelial cell biology to promote plaque formation.

Objective Endothelial cell activation drives early atherosclerotic plaque formation. Both fibronectin deposition and accumulation of oxidized LDL (oxLDL) occur early during atherogenesis and both are implicated in enhanced endothelial cell activation. However, interplay between these responses has not been established. The objective of our study was to determine whether endothelial matrix composition modulates the inflammatory properties of oxLDL. Approach and Results We now show that oxLDL-induced NF-κB activation, proinflammatory gene expression, and monocyte binding is significantly enhanced when endothelial cells are attached to fibronectin compared to basement membrane proteins. This enhanced response does not result from altered oxLDL receptor expression, oxLDL uptake, or reactive oxygen species production, but instead results from oxLDL-induced activation of the fibronectin-binding integrin α5β1. Preventing α5β1 signaling (blocking antibodies, knockout cells) inhibits oxLDL-induced NF-κB activation and VCAM-1 expression. Furthermore, oxLDL-drives α5β1-dependent integrin signaling through the focal adhesion kinase (FAK) pathway and FAK inhibition (PF-573228, siRNA) blunts oxLDL-induced NF-κB activation, VCAM-1 expression, and monocyte adhesion. Lastly, treatment with the α5β1 signaling inhibitor, ATN-161, significantly blunts atherosclerotic plaque development in ApoE deficient mice, characterized by reduced VCAM-1 expression and macrophage accumulation without affecting fibrous cap size. Conclusions Our data suggest that α5β1-mediated crosstalk between fibronectin and oxidized LDL regulates inflammation in early atherogenesis and therapeutics that inhibit α5 integrins may reduce inflammation without adversely affecting plaque structure.

The formation of atherosclerotic plaques in the large and medium sized arteries is classically driven by systemic factors, such as elevated cholesterol and blood pressure. However, work over the past several decades has established that atherosclerotic plaque development involves a complex coordination of both systemic and local cues that ultimately determine where plaques form and how plaques progress. While current therapeutics for atherosclerotic cardiovascular disease primarily target the systemic risk factors, a large array of studies suggest that the local microenvironment, including arterial mechanics, matrix remodeling, and lipid deposition, plays a vital role in regulating the local susceptibility to plaque development through the regulation of vascular cell function. Additionally, these microenvironmental stimuli are capable of tuning other aspects of the microenvironment through collective adaptation. In this review, we will discuss the components of the arterial microenvironment, how these components crosstalk to shape the local microenvironment, and the effect of microenvironmental stimuli on vascular cell function during atherosclerotic plaque formation.

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.

Background Atherosclerotic plaque formation results from chronic inflammation and fibroproliferative remodeling in the vascular wall. We previously demonstrated that both human and mouse atherosclerotic plaques show elevated expression of EphA2, a guidance molecule involved in cell-cell interactions and tumorigenesis. Methods Here, we assessed EphA2's role in atherosclerosis by deleting EphA2 in a mouse model of atherosclerosis (Apoe-/-) and by assessing EphA2 function in multiple vascular cell culture models. Following 8-16 weeks Western diet, male and female mice were assessed for atherosclerotic burden in the large vessels, and plasma lipid levels were analyzed. Results Despite enhanced weight gain and plasma lipid levels compared to Apoe-/- controls, EphA2-/-Apoe-/- knockout mice show diminished atherosclerotic plaque formation, characterized by reduced proinflammatory gene expression and plaque macrophage content. While plaque macrophages express EphA2, EphA2 deletion does not affect macrophage phenotype, inflammatory responses, and lipid uptake, and bone marrow chimeras suggest hematopoietic EphA2 deletion does not affect plaque formation. In contrast, endothelial EphA2 knockdown significantly reduces monocyte firm adhesion under flow. In addition, EphA2-/-Apoe-/- mice show reduced progression to advanced atherosclerotic plaques with diminished smooth muscle and collagen content. Consistent with this phenotype, EphA2 shows enhanced expression following smooth muscle transition to a synthetic phenotype, and EphA2 depletion reduces smooth muscle proliferation, mitogenic signaling, and extracellular matrix deposition both in atherosclerotic plaques and in vascular smooth muscle cells in culture. Conclusions Together these data identify a novel role for EphA2 in atherosclerosis, regulating both plaque inflammation and progression to advanced atherosclerotic lesions. Cell culture studies suggest that endothelial EphA2 contributes to atherosclerotic inflammation by promoting monocyte firm adhesion, whereas smooth muscle EphA2 expression may regulate the progression to advanced atherosclerosis by regulating smooth muscle proliferation and extracellular matrix deposition.

PAK2 mediates shear stress–induced NF-κB activation. Basement membrane proteins limit the proinflammatory response to shear by blocking the interaction of PAK2 with the adaptor protein Nck. This uncoupling response requires protein kinase A–dependent nitric oxide production and subsequent PAK2 phosphorylation on Ser-20 in the Nck-binding domain.

Objective: Alterations in extracellular matrix quantity and composition contribute to atherosclerosis, with remodeling of the subendothelial basement membrane to a fibronectin-rich matrix preceding lesion development. Endothelial cell interactions with fibronectin prime inflammatory responses to a variety of atherogenic stimuli; however, the mechanisms regulating early atherogenic fibronectin accumulation remain unknown. We previously demonstrated that oxidized LDL (oxLDL) promotes endothelial pro inflammatory gene expression by activating the integrin α5β1, a classic mediator of fibronectin fibrillogenesis. Approach and Results: We now show that oxLDL drives robust endothelial fibronectin deposition and inhibiting α5β1 (blocking antibodies, α5 knockout cells) completely inhibits oxLDL-induced fibronectin deposition. Consistent with this, inducible endothelial-specific α5 integrin deletion in ApoE knockout mice significantly reduces atherosclerotic plaque formation, associated with reduced early atherogenic inflammation. Unlike TGFβ-induced fibronectin deposition, oxLDL does not induce fibronectin expression (mRNA, protein) or the endothelial-to-mesenchymal transition phenotype. In addition, we show that cell-derived and plasma-derived fibronectin differentially affect endothelial function, with only cell-derived fibronectin capable of supporting oxLDL-induced VCAM-1 expression despite plasma fibronectin deposition by oxLDL. The inclusion of EIIIA and EIIIB domains in cell-derived fibronectin mediates this effect, as EIIIA/EIIIB knockout endothelial cells show diminished oxLDL-induced inflammation. Furthermore, our data suggests that EIIIA/EIIIB-positive cellular fibronectin is required for maximal α5β1 recruitment to focal adhesions and fibronectin fibrillogenesis. Conclusions: Taken together, our data demonstrate that endothelial α5 integrins drives oxLDL-induced fibronectin deposition and early atherogenic inflammation. Additionally, we show that α5β1-dependent endothelial fibronectin deposition mediates oxLDL-dependent endothelial inflammation and fibronectin fibrillogenesis.

Objective Altered subendothelial matrix composition regulates endothelial dysfunction and early atherosclerotic plaque formation. Hyperglycemia promotes endothelial matrix remodeling associated with multiple microvascular complications of diabetes, but a role for altered matrix composition in diabetic atherogenesis has not been described. Therefore, we sought to characterize the alterations in matrix composition during diabetic atherogenesis using both in vitro and in vivo model systems. Methods and Results Streptozotocin-induced diabetes in atherosclerosis-prone ApoE knockout mice promoted transitional matrix expression (fibronectin, thrombospondin-1) and deposition in intima of the aortic arch as determined by qRT-PCR array and immunohistochemistry. Early plaque formation occurs at discrete vascular sites exposed to disturbed blood flow patterns, whereas regions exposed to laminar flow are protected. Consistent with this pattern, hyperglycemia-induced transitional matrix deposition was restricted to regions of disturbed blood flow. Laminar flow significantly blunted high glucose-induced fibronectin expression (mRNA and protein) and fibronectin fibrillogenesis in endothelial cell culture models, whereas high glucose-induced fibronectin deposition was similar between disturbed flow and static conditions. Conclusions Taken together, these data demonstrate that flow patterns and hyperglycemia coordinately regulate subendothelial fibronectin deposition during early atherogenesis.