Mitochondrial Fission Promotes the Continued Clearance of Apoptotic Cells by Macrophages.

Clearance of apoptotic cells (ACs) by phagocytes (efferocytosis) prevents post-apoptotic necrosis and dampens inflammation. Defective efferocytosis drives important diseases, including atherosclerosis. For efficient efferocytosis, phagocytes must be able to internalize multiple ACs. We show here that uptake of multiple ACs by macrophages requires dynamin-related protein 1 (Drp1)-mediated mitochondrial fission, which is triggered by AC uptake. When mitochondrial fission is disabled, AC-induced increase in cytosolic calcium is blunted owing to mitochondrial calcium sequestration, and calcium-dependent phagosome formation around secondarily encountered ACs is impaired. These defects can be corrected by silencing the mitochondrial calcium uniporter (MCU). Mice lacking myeloid Drp1 showed defective efferocytosis and its pathologic consequences in the thymus after dexamethasone treatment and in advanced atherosclerotic lesions in fat-fed Ldlr-/- mice. Thus, mitochondrial fission in response to AC uptake is a critical process that enables macrophages to clear multiple ACs and to avoid the pathologic consequences of defective efferocytosis in vivo.

Regulatory T Cells Promote Macrophage Efferocytosis during Inflammation Resolution.

Regulatory T (Treg) cell responses and apoptotic cell clearance (efferocytosis) represent critical arms of the inflammation resolution response. We sought to determine whether these processes might be linked through Treg-cell-mediated enhancement of efferocytosis. In zymosan-induced peritonitis and lipopolysaccharide-induced lung injury, Treg cells increased early in resolution, and Treg cell depletion decreased efferocytosis. In advanced atherosclerosis, where defective efferocytosis drives disease progression, Treg cell expansion improved efferocytosis. Mechanistic studies revealed the following sequence: (1) Treg cells secreted interleukin-13 (IL-13), which stimulated IL-10 production in macrophages; (2) autocrine-paracrine signaling by IL-10 induced Vav1 in macrophages; and (3) Vav1 activated Rac1 to promote apoptotic cell engulfment. In summary, Treg cells promote macrophage efferocytosis during inflammation resolution via a transcellular signaling pathway that enhances apoptotic cell internalization. These findings suggest an expanded role of Treg cells in inflammation resolution and provide a mechanistic basis for Treg-cell-enhancement strategies for non-resolving inflammatory diseases.

Amino Acid Metabolism and Atherosclerotic Cardiovascular Disease.

Despite significant advances in medical treatments and drug development, atherosclerotic cardiovascular disease (ASCVD) remains a leading cause of death worldwide. Dysregulated lipid metabolism is a well-established driver of ASCVD. Unfortunately, even with potent lipid-lowering therapies, ASCVD-related deaths have continued to increase over the past decade, highlighting an incomplete understanding of the underlying risk factors and mechanisms of ASCVD. Accumulating evidence over the past decades indicates a correlation between amino acids and disease state. This review explores the emerging role of amino acid metabolism in ASCVD, uncovering novel potential biomarkers, causative factors, and therapeutic targets. Specifically, the significance of arginine and its related metabolites, homoarginine and polyamines, branched-chain amino acids, glycine, and aromatic amino acids, in ASCVD are discussed. These amino acids and their metabolites have been implicated in various processes characteristic of ASCVD, including impaired lipid metabolism, endothelial dysfunction, increased inflammatory response, and necrotic core development. Understanding the complex interplay between dysregulated amino acid metabolism and ASCVD provides new insights that may lead to the development of novel diagnostic and therapeutic approaches. Although further research is needed to uncover the precise mechanisms involved, it is evident that amino acid metabolism plays a role in ASCVD.

Loss of Macrophage mTORC2 Drives Atherosclerosis via FoxO1 and IL-1ß Signaling.

BACKGROUND: The mTOR (mechanistic target of rapamycin) pathway is a complex signaling cascade that regulates cellular growth, proliferation, metabolism, and survival. Although activation of mTOR signaling has been linked to atherosclerosis, its direct role in lesion progression and in plaque macrophages remains poorly understood. We previously demonstrated that mTORC1 (mTOR complex 1) activation promotes atherogenesis through inhibition of autophagy and increased apoptosis in macrophages. METHODS: Using macrophage-specific Rictor- and mTOR-deficient mice, we now dissect the distinct functions of mTORC2 pathways in atherogenesis. RESULTS: In contrast to the atheroprotective effect seen with blockade of macrophage mTORC1, macrophage-specific mTORC2-deficient mice exhibit an atherogenic phenotype, with larger, more complex lesions and increased cell death. In cultured macrophages, we show that mTORC2 signaling inhibits the FoxO1 (forkhead box protein O1) transcription factor, leading to suppression of proinflammatory pathways, especially the inflammasome/IL (interleukin)-1ß response, a key mediator of vascular inflammation and atherosclerosis. In addition, administration of FoxO1 inhibitors efficiently rescued the proinflammatory response caused by mTORC2 deficiency both in vitro and in vivo. Interestingly, collective deletion of macrophage mTOR, which ablates mTORC1- and mTORC2-dependent pathways, leads to minimal change in plaque size or complexity, reflecting the balanced yet opposing roles of these signaling arms. CONCLUSIONS: Our data provide the first mechanistic details of macrophage mTOR signaling in atherosclerosis and suggest that therapeutic measures aimed at modulating mTOR need to account for its dichotomous functions.

Spatially Resolved Metabolites in Stable and Unstable Human Atherosclerotic Plaques Identified by Mass Spectrometry Imaging.

BACKGROUND: Impairments in carbohydrate, lipid, and amino acid metabolism drive features of plaque instability. However, where these impairments occur within the atheroma remains largely unknown. Therefore, we sought to characterize the spatial distribution of metabolites within stable and unstable atherosclerosis in both the fibrous cap and necrotic core. METHODS: Atherosclerotic tissue specimens from 9 unmatched individuals were scored based on the Stary classification scale and subdivided into stable and unstable atheromas. After performing mass spectrometry imaging on these samples, we identified over 850 metabolite-related peaks. Using MetaboScape, METASPACE, and Human Metabolome Database, we confidently annotated 170 of these metabolites and found over 60 of these were different between stable and unstable atheromas. We then integrated these results with an RNA-sequencing data set comparing stable and unstable human atherosclerosis. RESULTS: Upon integrating our mass spectrometry imaging results with the RNA-sequencing data set, we discovered that pathways related to lipid metabolism and long-chain fatty acids were enriched in stable plaques, whereas reactive oxygen species, aromatic amino acid, and tryptophan metabolism were increased in unstable plaques. In addition, acylcarnitines and acylglycines were increased in stable plaques whereas tryptophan metabolites were enriched in unstable plaques. Evaluating spatial differences in stable plaques revealed lactic acid in the necrotic core, whereas pyruvic acid was elevated in the fibrous cap. In unstable plaques, 5-hydroxyindoleacetic acid was enriched in the fibrous cap. CONCLUSIONS: Our work here represents the first step to defining an atlas of metabolic pathways involved in plaque destabilization in human atherosclerosis. We anticipate this will be a valuable resource and open new avenues of research in cardiovascular disease.

Crosstalk Between Macrophages and Vascular Smooth Muscle Cells in Atherosclerotic Plaque Stability.

Most acute cardiovascular events are due to plaque rupture, with atheromas containing large necrotic cores and thin fibrous caps being more susceptible to rupture and lesions with small necrotic cores and thick fibrous caps being more protected from rupture. Atherosclerotic plaques are comprised various extracellular matrix proteins, modified lipoprotein particles, and cells of different origins, that is, vascular cells and leukocytes. Although much has been revealed about the mechanisms that lead to plaque instability, several key areas remain incompletely understood. This In-Focus Review highlights processes related to cellular crosstalk and the role of the tissue microenvironment in determining cell function and plaque stability. Recent advances highlight critical underpinnings of atherosclerotic plaque vulnerability, particularly impairments in the ability of macrophages to clear dead cells and phenotypic switching of vascular smooth muscle cells. However, these processes do not occur in isolation, as crosstalk between macrophages and vascular smooth muscle cells and interactions with their surrounding microenvironment play a significant role in determining plaque stability. Understanding these aspects of cellular crosstalk within an atherosclerotic plaque may shed light on how to modify cell behavior and identify novel approaches to transform rupture-prone atheromas into stable lesions.

SLAMF1 is expressed and secreted by hepatocytes and the liver in nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is one of the most prevalent forms of chronic liver disease in the United States and worldwide. Nonalcoholic steatohepatitis (NASH), the most advanced form of NAFLD, is characterized by hepatic steatosis associated with inflammation and hepatocyte death. No treatments are currently available for NASH other than lifestyle changes, and the disease lacks specific biomarkers. The signaling lymphocytic activation molecule family 1 (SLAMF1) protein is a self-ligand receptor that plays a role in orchestrating an immune response to some pathogens and cancers. We found that livers from humans and mice with NASH showed a more prominent immunohistochemistry staining for SLAMF1 than non-NASH controls. Furthermore, SLAMF1 levels are significantly increased in NASH plasma samples from mice and humans compared with their respective controls. In mice, the levels of SLAMF1 correlated significantly with the severity of the NASH phenotype. To test whether SLAMF 1 is expressed by hepatocytes, HepG2 cells and primary murine hepatocytes were treated with palmitic acid (PA) to induce a state of lipotoxicity mimicking NASH. We found that PA treatments of HepG2 cells and primary hepatocytes lead to significant increases in SLAMF1 levels. The downregulation of SLAMF1 in HepG2 cells improved the cell viability and reduced cytotoxicity. The in vivo data using mouse and human NASH samples suggests a potential role for this protein as a noninvasive biomarker for NASH. The in vitro data suggest a role for SLAMF1 as a potential therapeutic target to prevent hepatocyte death in response to lipotoxicity.NEW & NOTEWORTHY This study identified for the first time SLAMF1 as a mediator of hepatocyte death in nonalcoholic fatty liver disease (NASH) and as a marker of NASH in humans. There are no pharmacological treatments available for NASH, and diagnostic tools are limited to invasive liver biopsies. Therefore, since SLAMF1 levels correlate with disease progression and SLAMF1 mediates cytotoxic effects, this protein can be used as a therapeutic target and a clinical biomarker of NASH.

ODC (Ornithine Decarboxylase)-Dependent Putrescine Synthesis Maintains MerTK (MER Tyrosine-Protein Kinase) Expression to Drive Resolution.

OBJECTIVE: ODC (ornithine decarboxylase)-dependent putrescine synthesis promotes the successive clearance of apoptotic cells (ACs) by macrophages, contributing to inflammation resolution. However, it remains unknown whether ODC is required for other arms of the resolution program. Approach and Results: RNA sequencing of ODC-deficient macrophages exposed to ACs showed increases in mRNAs associated with heightened inflammation and decreases in mRNAs related to resolution and repair compared with WT (wild type) macrophages. In zymosan peritonitis, myeloid ODC deletion led to delayed clearance of neutrophils and a decrease in the proresolving cytokine, IL (interleukin)-10. Nanoparticle-mediated silencing of macrophage ODC in a model of atherosclerosis regression lowered IL-10 expression, decreased efferocytosis, enhanced necrotic core area, and reduced fibrous cap thickness. Mechanistically, ODC deletion lowered basal expression of MerTK (MER tyrosine-protein kinase)-an AC receptor-via a histone methylation-dependent transcriptional mechanism. Owing to lower basal MerTK, subsequent exposure to ACs resulted in lower MerTK-Erk (extracellular signal-regulated kinase) 1/2-dependent IL-10 production. Putrescine treatment of ODC-deficient macrophages restored the expression of both MerTK and AC-induced IL-10. CONCLUSIONS: These findings demonstrate that ODC-dependent putrescine synthesis in macrophages maintains a basal level of MerTK expression needed to optimally resolve inflammation upon subsequent AC exposure. Graphic Abstract: A graphic abstract is available for this article.

An ATF6-tPA pathway in hepatocytes contributes to systemic fibrinolysis and is repressed by DACH1.

Tissue-type plasminogen activator (tPA) is a major mediator of fibrinolysis and, thereby, prevents excessive coagulation without compromising hemostasis. Studies on tPA regulation have focused on its acute local release by vascular cells in response to injury or other stimuli. However, very little is known about sources, regulation, and fibrinolytic function of noninjury-induced systemic plasma tPA. We explore the role and regulation of hepatocyte-derived tPA as a source of basal plasma tPA activity and as a contributor to fibrinolysis after vascular injury. We show that hepatocyte tPA is downregulated by a pathway in which the corepressor DACH1 represses ATF6, which is an inducer of the tPA gene Plat Hepatocyte-DACH1-knockout mice show increases in liver Plat, circulating tPA, fibrinolytic activity, bleeding time, and time to thrombosis, which are reversed by silencing hepatocyte Plat Conversely, hepatocyte-ATF6-knockout mice show decreases in these parameters. The inverse correlation between DACH1 and ATF6/PLAT is conserved in human liver. These findings reveal a regulated pathway in hepatocytes that contributes to basal circulating levels of tPA and to fibrinolysis after vascular injury.

Endothelial FN (Fibronectin) Deposition by α5ß1 Integrins Drives Atherogenic Inflammation.

Objective- Alterations in extracellular matrix quantity and composition contribute to atherosclerosis, with remodeling of the subendothelial basement membrane to an FN (fibronectin)-rich matrix preceding lesion development. Endothelial cell interactions with FN prime inflammatory responses to a variety of atherogenic stimuli; however, the mechanisms regulating early atherogenic FN accumulation remain unknown. We previously demonstrated that oxLDL (oxidized low-density lipoprotein) promotes endothelial proinflammatory gene expression by activating the integrin α5ß1, a classic mediator of FN fibrillogenesis. Approach and Results- We now show that oxLDL drives robust endothelial FN deposition and inhibiting α5ß1 (blocking antibodies, α5 knockout cells) completely inhibits oxLDL-induced FN 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 TGFß (transforming growth factor ß)-induced FN deposition, oxLDL does not induce FN expression (mRNA, protein) or the endothelial-to-mesenchymal transition phenotype. In addition, we show that cell-derived and plasma-derived FN differentially affect endothelial function, with only cell-derived FN capable of supporting oxLDL-induced VCAM-1 (vascular cell adhesion molecule 1) expression, despite plasma FN deposition by oxLDL. The inclusion of alternative exon EIIIA (EDA) of FN (EIIIA) and alternative exon EIIIB (EDB) of FN (EIIIB) domains in cell-derived FN mediates this effect, as EIIIA/EIIIB knockout endothelial cells show diminished oxLDL-induced inflammation. Furthermore, our data suggest that EIIIA/EIIIB-positive cellular FN is required for maximal α5ß1 recruitment to focal adhesions and FN fibrillogenesis. Conclusions- Taken together, our data demonstrate that endothelial α5 integrins drive oxLDL-induced FN deposition and early atherogenic inflammation. Additionally, we show that α5ß1-dependent endothelial FN deposition mediates oxLDL-dependent endothelial inflammation and FN fibrillogenesis.

Cystathionine γ-Lyase Modulates Flow-Dependent Vascular Remodeling.

Objective- Flow patterns differentially regulate endothelial cell phenotype, with laminar flow promoting vasodilation and disturbed flow promoting endothelial proinflammatory activation. CSE (cystathionine γ-lyase), a major source of hydrogen sulfide (H2S) in endothelial cells, critically regulates cardiovascular function, by both promoting vasodilation and reducing endothelial activation. Therefore, we sought to investigate the role of CSE in the endothelial response to flow. Approach and Results- Wild-type C57Bl/6J and CSE knockout ( CSE-/-) mice underwent partial carotid ligation to induce disturbed flow in the left carotid. In addition, endothelial cells isolated from wild-type and CSE -/- mice were exposed to either laminar or oscillatory flow, an in vitro model of disturbed flow. Interestingly, laminar flow significantly reduced CSE expression in vitro, and only disturbed flow regions show discernable CSE protein expression in vivo, correlating with enhanced H2S production in wild-type C57BL/6J but not CSE-/- mice. Lack of CSE limited disturbed flow-induced proinflammatory gene expression (ICAM-1[intercellular adhesion molecule 1], VCAM-1 [vascular cell adhesion molecular 1]) and monocyte infiltration and CSE-/- endothelial cells showed reduced NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation and proinflammatory gene expression in response to oscillatory flow in vitro. In addition, CSE-/- mice showed reduced inward remodeling after partial carotid ligation. CSE-/- mice showed elevated vascular nitrite levels (measure of nitric oxide [NO]) in the unligated carotids, suggesting an elevation in baseline NO production, and the NO scavenger 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy-3-oxide normalized the reduced inward remodeling, but not inflammation, of ligated carotids in CSE-/- mice. Conclusions- CSE expression in disturbed flow regions critically regulates both endothelial activation and flow-dependent vascular remodeling, in part through altered NO availability.

The Type 1 Diabetes-Resistance Locus Idd22 Controls Trafficking of Autoreactive CTLs into the Pancreatic Islets of NOD Mice.

Type 1 diabetes (T1D) has a strong genetic component. The insulin dependent diabetes (Idd)22 locus was identified in crosses of T1D-susceptible NOD mice with the strongly T1D-resistant ALR strain. The NODcALR-(D8Mit293-D8Mit137)/Mx (NOD-Idd22) recombinant congenic mouse strain was generated in which NOD mice carry the full Idd22 confidence interval. NOD-Idd22 mice exhibit almost complete protection from spontaneous T1D and a significant reduction in insulitis. Our goal was to unravel the mode of Idd22-based protection using in vivo and in vitro models. We determined that Idd22 did not impact immune cell diabetogenicity or ß cell resistance to cytotoxicity in vitro. However, NOD-Idd22 mice were highly protected against adoptive transfer of T1D. Transferred CTLs trafficked to the pancreatic lymph node and proliferated to the same extent in NOD and NOD-Idd22 mice, yet the accumulation of pathogenic CTLs in the islets was significantly reduced in NOD-Idd22 mice, correlating with disease resistance. Pancreatic endothelial cells from NOD-Idd22 animals expressed lower levels of adhesion molecules, even in response to inflammatory stimuli. Lower adhesion molecule expression resulted in weaker adherence of T cells to NOD-Idd22 endothelium compared with NOD-derived endothelium. Taken together, these results provide evidence that Idd22 regulates the ability of ß cell-autoreactive T cells to traffic into the pancreatic islets and may represent a new target for pharmaceutical intervention to potentially prevent T1D.

EphA2 Expression Regulates Inflammation and Fibroproliferative Remodeling in Atherosclerosis.

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 the role of EphA2 in atherosclerosis by deleting EphA2 in a mouse model of atherosclerosis (Apoe-/-) and by assessing EphA2 function in multiple vascular cell culture models. After 8 to 16 weeks on a 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 with Apoe-/- controls, EphA2-/-Apoe-/- knockout mice show diminished atherosclerotic plaque formation, characterized by reduced proinflammatory gene expression and plaque macrophage content. Although plaque macrophages express EphA2, EphA2 deletion does not affect macrophage phenotype, inflammatory responses, and lipid uptake, and bone marrow chimeras suggest that 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 after 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.

Oxidized LDL induces FAK-dependent RSK signaling to drive NF-κB activation and VCAM-1 expression.

Oxidized low-density lipoprotein (oxLDL) accumulates early in atherosclerosis and promotes endothelial nuclear factor κB (NF-κB) activation, proinflammatory gene expression and monocyte adhesion. Like for other atherogenic factors, oxLDL-induced proinflammatory responses requires integrin-dependent focal adhesion kinase (FAK, also known as PTK2) signaling; however, the mechanism by which FAK mediates oxLDL-dependent NF-κB signaling has yet to be revealed. We now show that oxLDL induces NF-κB activation and VCAM-1 expression through FAK-dependent IκB kinase ß (IKKß, also known as IKBKB) activation. We further identify FAK-dependent activation of p90 ribosomal S6 kinase family proteins (RSK) as a crucial mediator of oxLDL-dependent IKKß and NF-κB signaling, as inhibiting RSK blocks oxLDL-induced IKKß and NF-κB activation, VCAM-1 expression and monocyte adhesion. Finally, transgenic mice containing a kinase-dead mutation in FAK specifically in the endothelial cells show reduced RSK activity, decreased VCAM-1 expression and reduced macrophage accumulation in regions of early atherosclerosis. Taken together, our data elucidates a new mechanism whereby oxLDL-induced endothelial FAK signaling drives an ERK-RSK pathway to activate IKKß and NF-κB signaling and proinflammatory gene expression.

The arterial microenvironment: the where and why of atherosclerosis.

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. Although 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 remodelling 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 cross-talk to shape the local microenvironment, and the effect of microenvironmental stimuli on vascular cell function during atherosclerotic plaque formation.

αvß3 Integrins Mediate Flow-Induced NF-κB Activation, Proinflammatory Gene Expression, and Early Atherogenic Inflammation.

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 α5ß1, αvß3, and αvß5. 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 αvß3 integrins (S247, siRNA), but not α5ß1 or αvß5, blunts shear stress-induced proinflammatory signaling (NF-κB, p21-activated kinase) and gene expression (ICAM1, VCAM1). Importantly, inhibiting αvß3 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 αv integrins (S247), but not α5 (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 αv integrin deletion similarly blunted inflammation in models of disturbed flow and diet-induced atherogenesis but did not affect smooth muscle incorporation. Our studies identify αvß3 as the primary integrin heterodimer mediating shear stress-induced proinflammatory responses and as a key contributor to early atherogenic inflammation.

α5ß1 integrin signaling mediates oxidized low-density lipoprotein-induced inflammation and early atherosclerosis.

OBJECTIVE: Endothelial cell activation drives early atherosclerotic plaque formation. Both fibronectin deposition and accumulation of oxidized low-density lipoprotein (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 nuclear factor-κB activation, proinflammatory gene expression, and monocyte binding are significantly enhanced when endothelial cells are attached to fibronectin compared with basement membrane proteins. This enhanced response does not result from altered oxLDL receptor expression, oxLDL uptake, or reactive oxygen species production, but results from oxLDL-induced activation of the fibronectin-binding integrin α5ß1. Preventing α5ß1 signaling (blocking antibodies, knockout cells) inhibits oxLDL-induced nuclear factor-κB activation and vascular cell adhesion molecule-1 expression. Furthermore, oxLDL drives α5ß1-dependent integrin signaling through the focal adhesion kinase pathway, and focal adhesion kinase inhibition (PF-573228, small interfering RNA) blunts oxLDL-induced nuclear factor-κB activation, vascular cell adhesion molecule-1 expression, and monocyte adhesion. Last, treatment with the α5ß1 signaling inhibitor, ATN-161, significantly blunts atherosclerotic plaque development in apolipoprotein E-deficient mice, characterized by reduced vascular cell adhesion molecule-1 expression and macrophage accumulation without affecting fibrous cap size. CONCLUSIONS: Our data suggest that α5ß1-mediated cross-talk between fibronectin and oxLDL regulates inflammation in early atherogenesis and that therapeutics that inhibit α5 integrins may reduce inflammation without adversely affecting plaque structure.

Resveratrol promotes endothelial cell wound healing under laminar shear stress through an estrogen receptor-α-dependent pathway.

Restenosis is an adverse outcome of angioplasty, characterized by vascular smooth muscle cell (VSMC) hyperplasia. However, therapies targeting VSMC proliferation delay re-endothelialization, increasing the risk of thrombosis. Resveratrol (RESV) inhibits restenosis and promotes re-endothelialization after arterial injury, but in vitro studies assessing RESV-mediated effects on endothelial cell growth contradict these findings. We thus hypothesized that fluid shear stress, mimicking physiological blood flow, would recapitulate RESV-dependent endothelial cell wound healing. Since RESV is an estrogen receptor (ER) agonist, we tested whether RESV promotes re-endothelialization through an ER-α-dependent mechanism. Mice fed a high-fat diet or a diet supplemented with RESV were subjected to carotid artery injury. At 7 days after injury, RESV significantly accelerated re-endothelialization compared with vehicle. In vitro wound healing assays demonstrated that RESV exhibits cell-type selectivity, inhibiting VSMC, but not endothelial cell growth. Under laminar shear stress (LSS), RESV dramatically enhanced endothelial cell wound healing and increased both the activation of extracellular signal-regulated kinase (ERK) and endothelial cell proliferation. Under LSS, small interfering RNA against ER-α, but not endothelial nitric oxide synthase, abolished RESV-induced ERK activation, endothelial cell proliferation, and wound healing. Thus these studies suggest that the EC phenotype induced by LSS better models the prohealing effects of RESV and that RESV and LSS interact to promote an ER-α-dependent mitogenic effect in endothelial cells.

Dysregulated cellular metabolism in atherosclerosis: mediators and therapeutic opportunities.

Accumulating evidence over the past decades has revealed an intricate relationship between dysregulation of cellular metabolism and the progression of atherosclerotic cardiovascular disease. However, an integrated understanding of dysregulated cellular metabolism in atherosclerotic cardiovascular disease and its potential value as a therapeutic target is missing. In this Review, we (1) summarize recent advances concerning the role of metabolic dysregulation during atherosclerosis progression in lesional cells, including endothelial cells, vascular smooth muscle cells, macrophages and T cells; (2) explore the complexity of metabolic cross-talk between these lesional cells; (3) highlight emerging technologies that promise to illuminate unknown aspects of metabolism in atherosclerosis; and (4) suggest strategies for targeting these underexplored metabolic alterations to mitigate atherosclerosis progression and stabilize rupture-prone atheromas with a potential new generation of cardiovascular therapeutics.