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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., Competing Interests: Disclosures None.

While the opioid epidemic has garnered significant attention, the use of methamphetamines is growing worldwide independent of wealth or region. Following overdose and accidents, the leading cause of death in methamphetamine users is cardiovascular disease, because of significant effects of methamphetamine on vasoconstriction, pulmonary hypertension, atherosclerotic plaque formation, cardiac arrhythmias, and cardiomyopathy. In this review, we examine the current literature on methamphetamine-induced changes in cardiovascular health, discuss the potential mechanisms regulating these varied effects, and highlight our deficiencies in understanding how to treat methamphetamine-associated cardiovascular dysfunction.

Objective- IL (interleukin)-33 has been shown to play a role in endothelial dysfunction, but its role in atherosclerosis is controversial. Therefore, the purpose of this study is to examine its role in vascular wall remodeling following injury. Approach and Results- Thrombin induced IL-33 expression in a time-dependent manner in human aortic smooth muscle cells and inhibition of its activity by its neutralizing antibody suppressed thrombin induced human aortic smooth muscle cell migration but not DNA synthesis. In exploring the mechanisms, we found that Par1 (protease-activated receptor 1), Gαq/11 (Gα protein q/11), PLCβ3 (phospholipase Cβ3), NFATc1 (nuclear factor of activated T cells), E2F1 (E2F transcription factor 1), and LMCD1 (LIM and cysteine-rich domains protein 1) are involved in thrombin-induced IL-33 expression and migration. Furthermore, we identified an NFAT-binding site at -100 nt that mediates thrombin-induced IL-33 promoter activity. Interestingly, we observed that NFATc1, E2F1, and LMCD1 bind to NFAT site in response to thrombin and found that LMCD1, while alone has no significant effect, enhanced either NFATc1 or E2F1-dependent IL-33 promoter activity. In addition, we found that guidewire injury induces IL-33 expression in SMC and its neutralizing antibodies substantially reduce SMC migration and neointimal growth in vivo. Increased expression of IL-33 was also observed in human atherosclerotic lesions as compared to arteries without any lesions. Conclusions- The above findings reveal for the first time that thrombin-induced human aortic smooth muscle cell migration and injury-induced neointimal growth require IL-33 expression. In addition, thrombin-induced IL-33 expression requires LMCD1 enhanced combinatorial activation of NFATc1 and E2F1.

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 α5β1, a classic mediator of FN fibrillogenesis. Approach and Results- We now show that oxLDL drives robust endothelial FN deposition and inhibiting α5β1 (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 TGFβ (transforming growth factor β)-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 α5β1 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 α5β1-dependent endothelial FN deposition mediates oxLDL-dependent endothelial inflammation and FN fibrillogenesis.

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 (H 2 S) 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 H 2 S 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.

Objective: Macrophage proinflammatory responses induced by modified low-density lipoproteins (modLDL) contribute to atherosclerotic progression. How modLDL causes macrophages to become proinflammatory is still enigmatic. Macrophage foam cell formation induced by modLDL requires glycerolipid synthesis. Lipin-1, a key enzyme in the glycerolipid synthesis pathway, contributes to modLDL-elicited macrophage proinflammatory responses in vitro. The objective of this study was to determine whether macrophage-associated lipin-1 contributes to atherogenesis and to assess its role in modLDL-mediated signaling in macrophages., Approach and Results: We developed mice lacking lipin-1 in myeloid-derived cells and used adeno-associated viral vector 8 expressing the gain-of-function mutation of mouse proprotein convertase subtilisin/kexin type 9 (adeno-associated viral vector 8-proprotein convertase subtilisin/kexin type 9) to induce hypercholesterolemia and plaque formation. Mice lacking myeloid-associated lipin-1 had reduced atherosclerotic burden compared with control mice despite similar plasma lipid levels. Stimulation of bone marrow-derived macrophages with modLDL activated a persistent protein kinase Cα/βII-extracellular receptor kinase1/2-jun proto-oncogene signaling cascade that contributed to macrophage proinflammatory responses that was dependent on lipin-1 enzymatic activity., Conclusions: Our data demonstrate that macrophage-associated lipin-1 is atherogenic, likely through persistent activation of a protein kinase Cα/βII-extracellular receptor kinase1/2-jun proto-oncogene signaling cascade that contributes to foam cell proinflammatory responses. Taken together, these results suggest that modLDL-induced foam cell formation and modLDL-induced macrophage proinflammatory responses are not independent consequences of modLDL stimulation but rather are both directly influenced by enhanced lipid synthesis., (© 2017 American Heart Association, Inc.)

Objective: Neutrophil recruitment is a key process in the pathogenesis of stroke, and may provide a valuable therapeutic target. Targeting the melanocortin (MC) receptors has previously shown to inhibit leukocyte recruitment in peripheral inflammation, however, it is not known whether treatments are effective in the unique cerebral microvascular environment. Here, we provide novel research highlighting the effects of the MC peptides on cerebral neutrophil recruitment, demonstrating important yet discrete roles for both MC1 and MC3., Approach and Results: Using intravital microscopy, in 2 distinct murine models of cerebral ischemia-reperfusion (I/R) injury, we have investigated MC control for neutrophil recruitment. After global I/R, pharmacological treatments suppressed pathological neutrophil recruitment. MC1 selective treatment rapidly inhibited neutrophil recruitment while a nonselective MC agonist provided protection even when coadministered with an MC3/4 antagonist, suggesting the importance of early MC1 signaling. However, by 2-hour reperfusion, MC1-mediated effects were reduced, and MC3 anti-inflammatory circuits predominated. Mice bearing a nonfunctional MC1 displayed a transient exacerbation of neutrophil recruitment after global I/R, which diminished by 2 hours. However importantly, enhanced inflammatory responses in both MC1 mutant and MC3 (-/-) mice resulted in increased infarct size and poor functional outcome after focal I/R. Furthermore, we used an in vitro model of leukocyte recruitment to demonstrate these anti-inflammatory actions are also effective in human cells., Conclusions: These studies reveal for the first time MC control for neutrophil recruitment in the unique pathophysiological context of cerebral I/R, while also demonstrating the potential therapeutic value of targeting multiple MCs in developing effective therapeutics., (© 2015 American Heart Association, Inc.)

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 α5β1. Preventing α5β1 signaling (blocking antibodies, knockout cells) inhibits oxLDL-induced nuclear factor-κB activation and vascular cell adhesion molecule-1 expression. Furthermore, oxLDL drives α5β1-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 α5β1 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 α5β1-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., (© 2014 American Heart Association, Inc.)

Objective: Interleukin-19 (IL-19) is a putative Th2, anti-inflammatory interleukin. Its expression and potential role in atherogenesis are unknown. IL-19 is not detected in normal artery and is expressed to a greater degree in plaque from symptomatic versus asymptomatic patients, suggesting a compensatory counter-regulatory function. We tested whether IL-19 could reduce atherosclerosis in susceptible mice and identified plausible mechanisms., Approach and Results: LDLR(-/-) mice fed an atherogenic diet and injected with either 1.0 or 10.0 ng/g per day recombinant mouse IL-19 had significantly less plaque area in the aortic arch compared with controls (P<0.0001). Weight gain, cholesterol, and triglyceride levels were not significantly different. Gene expression in splenocytes from IL-19-treated mice demonstrated immune cell Th2 polarization, with decreased expression of T-bet, interferon-γ, interleukin-1β, and interleukin-12β and increased expression of GATA3 and FoxP3 mRNA. A greater percentage of lymphocytes were Th2 polarized in IL-19-treated mice. Cellular characterization of plaque by immunohistochemistry demonstrated that IL-19-treated mice have significantly less macrophage infiltrate compared with controls (P<0.001). Intravital microscopy revealed significantly less leukocyte adhesion in wild-type mice injected with IL-19 and fed an atherogenic diet compared with controls. Treatment of cultured endothelial cells, vascular smooth muscle cells, and bone marrow-derived macrophages with IL-19 resulted in a significant decrease in chemokine mRNA and mRNA stability protein human antigen R., Conclusions: These data suggest that IL-19 is a potent inhibitor of experimental atherosclerosis, with diverse mechanisms including immune cell polarization, decrease in macrophage adhesion, and decrease in gene expression. This may identify IL-19 as a novel therapeutic to limit vascular inflammation.

Objective: Upregulated expression of endothelial adhesion molecules and subsequent binding to cognate monocytic receptors are established paradigms in atherosclerosis. However, these proteins are the scaffolds, with their posttranslational modification with sugars providing the actual ligands. We recently showed that tumor necrosis factor-α increased hypoglycosylated (mannose-rich) N-glycans on the endothelial surface. In the present study, our aim was to determine whether (1) hypoglycosylated N-glycans are upregulated by proatherogenic stimuli (oscillatory flow) in vitro and in vivo, and (2) mannose residues on hypoglycosylated endothelial N-glycans mediate monocyte rolling and adhesion., Methods and Results: Staining with the mannose-specific lectins concanavalin A and lens culinaris agglutinin was increased in human aortic endothelial cells exposed to oscillatory shear or tumor necrosis factor-α and at sites of plaque development and progression in both mice and human vessels. Increasing surface N-linked mannose by inhibiting N-glycan processing potentiated monocyte adhesion under flow during tumor necrosis factor-α stimulation. Conversely, enzymatic removal of high-mannose N-glycans, or masking mannose residues with lectins, significantly decreased monocyte adhesion under flow. These effects occurred without altering induced expression of adhesion molecule proteins., Conclusions: Hypoglycosylated (high mannose) N-glycans are present on the endothelial cell surface at sites of early human lesion development and are novel effectors of monocyte adhesion during atherogenesis.

Objective: Endothelial cell activation results in altered cell-cell interactions with adjacent endothelial cells and with infiltrating leukocytes. Eph receptors and their ephrin ligands regulate cell-cell interactions during tissue remodeling, and multiple proinflammatory mediators induce endothelial EphA receptor and ephrinA ligand expression. Therefore, we sought to elucidate the role of EphA receptors and ephrinA ligands in endothelial cell activation and atherosclerosis., Methods and Results: Quantitative reverse transcription-polymerase chain reaction screening for EphA/ephrinA expression in atherosclerosis-prone macrovascular endothelium identified EphA2, EphA4, and ephrinA1 as the dominant isoforms. Endothelial activation with oxidized low-density lipoprotein and proinflammatory cytokines induced EphA2 and ephrinA1 expression and sustained EphA2 activation, whereas EphA4 expression was unaffected. Atherosclerotic plaques from mice and humans showed enhanced EphA2 and ephrinA1 expression colocalizing in the endothelial cell layer. EphA2 activation with recombinant Fc-ephrinA1 induced proinflammatory gene expression (eg vascular cell adhesion molecule-1, E-selectin) and stimulated monocyte adhesion, whereas inhibiting EphA2 (small interfering RNA, pharmacological inhibitors) abrogated both ephrinA1-induced and oxidized low-density lipoprotein-induced vascular cell adhesion molecule-1 expression., Conclusion: The current data suggest that enhanced EphA2 signaling during endothelial cell activation perpetuates proinflammatory gene expression. Coupled with EphA2 expression in mouse and human atherosclerotic plaques, these data implicate EphA2 as a novel proinflammatory mediator and potential regulator of atherosclerotic plaque development.

Objective: Smooth muscle cell (SMC) phenotypic modulation, an important component of atherosclerosis progression, is critically regulated by the matrix, with normal components of the healthy SMC matrix limiting modulation and atherosclerosis-associated transitional matrix proteins promoting phenotypic modulation. We sought to determine how collagen IV (which comprises the healthy artery wall) and monomeric collagen I (which comprises atherosclerotic lesions) differentially affect SMC phenotype., Methods and Results: Plating SMCs on collagen IV resulted in elevated expression of SMC contractility proteins compared to collagen I. Concurrent with enhanced contractile gene expression, collagen IV stimulates binding of SRF to CArG boxes in the promoters of smooth muscle actin and smooth muscle myosin heavy chain. Coll IV also stimulated the expression of myocardin, a critical SRF coactivator required to drive expression of SMC specific genes. In contrast to collagen IV, collagen I stimulated enhanced expression of the inflammatory protein vascular cell adhesion molecule (VCAM)-1. NF-kappaB and NFAT-binding sites in the VCAM-1 promoter are critical for collagen I-mediated expression of VCAM-1 promoter activity. However, only inhibitors of NFAT, not NF-kappaB, were able to reduce collagen I-associated VCAM expression, and collagen I but not collagen IV stimulated NFAT transcriptional activity., Conclusions: These results show for the first time that collagen IV and collagen I differentially affect smooth muscle phenotypic modulation through multiple pathways.

Background: 12/15-lipoxygenase (12/15-LO) activity leads to the production of the proinflammatory eicosanoids 12-S-hydroxyeicosatetraenoic acid (12SHETE) and 13-S-hydroxyoctadecadienoic acid. We have previously shown a 3.5-fold increase in endothelial intercellular adhesion molecule (ICAM)-1 expression in mice overexpressing the 12/15-LO gene. We examined whether 12/15-LO activity regulated endothelial ICAM-1 expression., Methods and Results: Freshly isolated aortic endothelial cells (EC) from 12/15-LO transgenic mice had significantly greater nuclear factor-kappaB (NF-kappaB) activation and ICAM mRNA expression compared with C57BL/6J control. 12/15-LO transgenic EC showed elevated RhoA activity, and inhibition of RhoA using either C3 toxin or the Rho-kinase inhibitor Y-27632 blocked NF-kappaB activation, ICAM-1 induction, and monocyte adhesion. Furthermore, we show that 12SHETE activates protein kinase Calpha, which forms a complex with active RhoA and is required for NF-kappaB-dependent ICAM expression in response to 12SHETE., Conclusions: The 12/15-LO pathway stimulates ICAM-1 expression through the RhoA/protein kinase Calpha-dependent activation of NF-kappaB. These findings identify a major signaling pathway in EC through which 12/15-LO contributes to vascular inflammation and atherosclerosis.