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.

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.

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.

Previous research has shown that podocytes unable to assemble heparan sulfate on cell surface proteoglycan core proteins have compromised cell-matrix interactions. This report further explores the role of N-sulfation of intact heparan chains in podocyte-matrix interactions. For the purposes of this study, a murine model in which the enzyme N-deacetylase/N-sulfotransferase 1 (NDST1) was specifically deleted in podocytes and immortalized podocyte cell lines lacking NDST1 were developed and used to explore the effects of such a mutation on podocyte behavior in vitro. NDST1 is a bifunctional enzyme, ultimately responsible for N-sulfation of heparan glycosaminoglycans produced by cells. Immunostaining of glomeruli from mice whose podocytes were null for Ndst1 (Ndst1(-/-)) showed a disrupted pattern of localization for the cell surface proteoglycan, syndecan-4, and for α-actinin-4 compared with controls. The pattern of immunostaining for synaptopodin and nephrin did not show as significant alterations. In vitro studies showed that Ndst1(-/-) podocytes attached, spread, and migrated less efficiently than Ndst1(+/+) podocytes. Immunostaining in vitro for several markers for molecules involved in cell-matrix interactions showed that Ndst1(-/-) cells had decreased clustering of syndecan-4 and decreased recruitment of protein kinase-Cα, α-actinin-4, vinculin, and phospho-focal adhesion kinase to focal adhesions. Total intracellular phospho-focal adhesion kinase was decreased in Ndst1(-/-) compared with Ndst1(+/+) cells. A significant decrease in the abundance of activated integrin α5β1 on the cell surface of Ndst1(-/-) cells compared with Ndst1(+/+) cells was observed. These results serve to highlight the critical role of heparan sulfate N-sulfation in facilitating normal podocyte-matrix interactions.

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. 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. Correspondingly, oxLDL activates focal adhesion kinase (FAK), the downstream signaling partner to integrin α5β1. We also found enhanced integrin α5 expression and active FAK in the endothelium overlying atherosclerotic plaques in both mice and humans. Preventing α5β1 signaling (blocking antibodies, knockout cells) inhibits oxLDL-induced NF-κB activation and VCAM-1 expression. Also, inhibiting FAK kinase activity utilizing a small molecule inhibitor (PF-573228), siRNA, and kinase dead fibroblasts reduces oxLDL endothelial cell activation. Furthermore, treatment with the α5β1 signaling inhibitor, ATN-161, significantly blunts atherosclerotic plaque development in ApoE deficient mice, characterized by reduced macrophage accumulation without affecting fibrous cap size. Therefore, our data suggest that α5β1-mediated crosstalk between fibronectin and oxidized LDL regulates inflammation in early atherogenesis.

Rational The frictional shear stress generated by blood flow activates integrins on the endothelial cell surface, and signaling downstream of newly activated integrins regulates several flow-induced responses including inflammation and permeability. Signaling through transitional matrix binding integrins (α5β1, αvβ3 and αvβ5) drives shear stress-induced NF-κB activation and proinflammatory gene expression; however the specific contribution of each of these integrins remains unclear. Objective To address which transitional matrix-binding integrin mediates shear stress-induced endothelial cell. Methods and Results We now show that the small molecule αv integrin inhibitor S247, but not the α5 inhibitor ATN-161, abolished flow-induced proinflammatory signaling (PAK, NF-κB). Blocking αv expression using siRNA similarly prevents flow-induced activation of PAK and NF-κB, whereas siRNA-mediated knock-down of α5 did not. Furthermore, αv and β3 knockdown but not α5 and β5 knockdown inhibited flow-induced ICAM-1 and VCAM-1 expression (both mRNA and protein) following either short term (5 hr) laminar and long term oscillatory (24 hr) shear stress, indicating αvβ3 is the mediator for flow induced endothelial cell inflammation. In contrast, αv knockdown did not affect TNFα-induced ICAM-1 and VCAM-1 expression, suggesting the anti-inflammatory effect of αv inhibition is specific to shear stress. Integrin inhibition did not affect all shear stress responses, as neither α5 nor αv siRNA affected shear stress-induced extracellular-signal regulated kinase (ERK1/2) activation. Lastly, inhibiting αv integrins using the small molecule inhibitor S247 reduces atherosclerotic plaque formation in ApoE knockout mice. Conclusion Taken together, these studies suggest that αvβ3 integrins mediate shear stress-induced proinflammatory responses associated with the transitional matrix in vitro and early atherosclerotic plaque formation in vivo .

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. Published data from our lab and others demonstrate that endothelial cell interactions with fibronectin prime inflammatory responses to a variety of atherogenic stimuli. However, the mechanisms regulating early atherogenic fibronectin accumulation remain unknown. Work from our group previously demonstrated that oxidized LDL (oxLDL) promotes endothelial proinflammatory gene expression by activating the integrin α5β1, a classic mediator of fibronectin fibrillogenesis. We now show that treating endothelial cells with oxLDL induces fibronectin deposition and inhibiting α5β1 (blocking antibodies, α5 knockout cells) completely inhibits oxLDL-induced fibronectin deposition. While endothelial fibronectin expression remains unchanged, oxLDL robustly stimulates the deposition of endothelial cell-derived fibronectin associated with a significant reduction in intracellular fibronectin. Interestingly, loss of endothelial cell-derived fibronectin, but not plasma fibronectin, prevents integrin α5 localization to focal adhesions, reduces fibronectin fibril length, and inhibits oxLDL-induced VCAM-1 expression. In addition, inducible endothelial-specific deletion of α5 integrins significantly blunts atherosclerotic plaque formation in ApoE knockout mice, suggesting an important role for this integrin in early endothelial activation. Taken together, our data demonstrate that oxLDL stimulates α5 integrin-dependent subendothelial matrix remodeling and endothelial proinflammatory gene expression through the deposition of fibronectin.

Podocytes adhere to the glomerular basement membrane via cell surface receptors which include heparan sulfate (HS) proteoglycans (PG). Podocytes unable to assemble HS glycosaminoglycan chains have defective cell attachment and spreading, and cytoskeletal disruption in vitro and in vivo. The current report explores the importance of heparan sulfation in regulating podocyte cell‐matrix interactions, using an immortalized podocyte cell line having the genomic deletion of N‐deacetylase/ N‐sulfotransferase 1 (NDST1), the enzyme responsible for N‐sulfation of HS. Compared to wild type podocytes, mutant podocytes attached, spread and migrated less efficiently in assays than control cells. In the process of cell‐matrix interaction, the mutant cells showed decreased clustering of syndecan 4, the cell surface HSPG involved in focal adhesion formation, and decreased recruitment of PKCα, α‐actinin 4, vinculin, and pFAK to focal adhesions. Cell surface integrin expression and integrin activation/signaling was significantly diminished in the mutant cells. These results serve to highlight the critical role of HS N‐sulfation in regulating the ability of podocytes to interact with the extracellular matrix.Grant Funding Source: NIDDK RO1‐DK07786