Non-canonical NF-κB contributes to endothelial pyroptosis and atherogenesis dependent on IRF-1.

Cell inflammation and death are closely linked processes contributing to endothelial dysfunction, which plays a critical role in atherogenesis. Activation of the NLRP3 inflammasome causes pyroptosis, the Gasdermin D (GSDMD)-mediated inflammatory cell death. The non-canonical NF-κB pathway has been implicated in inflammation; however, its role in NLRP3 inflammasome-mediated endothelial dysfunction has not been investigated. This study investigated a role for the non-canonical NF-κB pathway in regulating endothelial pyroptosis as it relates to atherogenesis. Immunohistochemistry indicated inflammasome activation in the endothelial cells (EC) of human atherosclerotic arteries. Flow cytometry and Western blot analysis revealed that oxidized low-density lipoprotein (oxLDL) activated the NLRP3 inflammasome, concomitant with the activation of non-canonical NF-κB in primary human aortic EC. Interference of NF-κB inducing kinase (NIK), the key regulator of the non-canonical pathway, significantly attenuated oxLDL- or LPS/ATP-induced NLRP3 inflammasome activation, pyroptosis, IL-1ß, and IL-18 secretion. In contrast, overexpression of NIK exacerbated these responses. Chromatin immunoprecipitation revealed that activation of the non-canonical NF-κB pathway upregulated the transcription factor IRF-1 through RelB/p52 binding to its promoter region at -782/-770. In addition to the known target CASP1, RNA sequencing further identified GSDMD as a target gene of IRF-1. IRF-1 but not RelB/p52 interacted with the GSDMD promoter at -526/-515 and the CASP1 promoter at -11/10 to promote the expression and CASP1-mediated activation of GSDMD. Consistent with the observations in cultured endothelium, endothelial-specific deficiency of NIK or IRF-1 attenuated atherosclerosis in high-fat diet-fed Apoe-null mice. These data demonstrate that the non-canonical NF-κB pathway contributes to NLRP3 inflammasome-mediated endothelial pyroptosis and the development of atherosclerosis through GSDMD activation in a manner dependent on IRF-1. Further investigation may facilitate the identification of specific therapeutic targets for atherosclerotic heart diseases.

mTOR contributes to endothelium-dependent vasorelaxation by promoting eNOS expression and preventing eNOS uncoupling.

Clinically used inhibitors of mammalian target of rapamycin (mTOR) negatively impacts endothelial-dependent vasodilatation (EDD) through unidentified mechanisms. Here we show that either the endothelium-specific deletion of Mtor to inhibit both mTOR complexes, or depletion of Raptor or Rictor to disrupt mTORC1 or mTORC2, causes impaired EDD, accompanied by reduced NO in the serum of mice. Consistently, inhibition of mTOR decreases NO production by human and mouse EC. Specifically, inhibition of mTORC1 suppresses eNOS gene expression, due to impairment in p70S6K-mediated posttranscriptional regulation of the transcription factor KLF2 expression. In contrast to mTORC1 inhibition, a positive-feedback between MAPK (p38 and JNK) activation and Nox2 upregulation contributes to the excessive generation of reactive oxygen species (ROS), which causes eNOS uncoupling and decreased NO bioavailability in mTORC2-inhibited EC. Adeno-associated virus-mediated EC-specific overexpression of KLF2 or suppression of Nox2 restores EDD function in endothelial mTORC1- or mTORC2-inhibited mice.

Targeting the KCa3.1 channel suppresses diabetes-associated atherosclerosis via the STAT3/CD36 axis.

BACKGROUND: In diet-induced arterial atherosclerosis, increased KCa3.1 channel was associated with atherosclerotic plaque progression and instability. Macrophages are involved in the formation of atherosclerotic plaques, and the release of inflammatory cytokines and oxygen free radicals promotes plaque progression. However, whether the macrophage KCa3.1 channel facilitates diabetes-accelerated atherosclerosis is still unclear. This study investigated atherosclerotic plaque in ApoE-/- mice regulated by the KCa3.1 channel. METHODS AND RESULTS: In vivo, blocking KCa3.1channel inhibit the development of the atherosclerotic lesion in diabetic ApoE-/- mice fed with a high-fat diet. In vitro, upregulation of KCa3.1 channel level occurred in RAW264.7 cells treated with HG plus ox-LDL in a time-dependent manner. Blocking KCa3.1 significantly reduced the uptake of ox-LDL in mice peritoneal macrophages. Further studies indicated the KCa3.1 siRNA and TRAM-34 (KCa3.1 inhibitor) attenuated the scavenger receptor CD36 expression via inhibiting STAT3 phosphorylation. CONCLUSION: Blockade of macrophage KCa3.1 channel inhibit cellular oxidized low-density lipoprotein accumulation and decrease proinflammation factors expression via STAT3/CD36 axis. This study provided a novel therapeutic target to reduce the risk of atherosclerosis development in diabetic patients.

mTOR Inhibition Promotes Pneumonitis through Inducing Endothelial Contraction and Hyperpermeability.

Compromised endothelial-cell (EC) barrier function is a hallmark of inflammatory diseases. mTOR inhibitors, widely applied as clinical therapies, cause pneumonitis through mechanisms that are not yet fully understood. This study aimed to elucidate the EC mechanisms underlying the pathogenesis of pneumonitis caused by mTOR inhibition (mTORi). Mice with EC-specific deletion of mTOR complex components (Mtor, Rptor or Rictor) were administered LPS to induce pulmonary injury. Cultured ECs were treated with pharmacologic inhibitors, siRNA, or overexpression plasmids. EC barrier function was evaluated in vivo with Evans blue assay and in vitro by measurement of transendothelial electrical resistance and albumin flux. mTORi increased basal and TNFα-induced EC permeability, which was caused by myosin light chain (MLC) phosphorylation-dependent cell contraction. Inactivation of mTOR kinase activity by mTORi triggered PKCδ/p38/NF-κB signaling that significantly upregulated TNFα-induced MLCK (MLC kinase) expression, whereas Raptor promoted the phosphorylation of PKCα/MYPT1 independently of its interaction with mTOR, leading to suppression of MLCP (MLC phosphatase) activity. EC-specific deficiency in mTOR, Raptor or Rictor aggravated lung inflammation in LPS-treated mice. These findings reveal that mTORi induces PKC-dependent endothelial MLC phosphorylation, contraction, and hyperpermeability that promote pneumonitis.

Effects of cranberry beverages on oxidative stress and gut microbiota in subjects with Helicobacter pylori infection: a randomized, double-blind, placebo-controlled trial.

Helicobacter pylori-induced oxidative stress plays an important role in gastric diseases. H. pylori disturbs gut microbiota. The objective is to investigate the effects of cranberry beverages on oxidative stress biomarkers and gut microbiota in H. pylori positive subjects. 171 H. pylori positive participants were randomly assigned to one of the three groups: high-dose (HCb; 480 mL cranberry beverage), low-dose (LCb; 240 mL cranberry beverage plus 240 mL placebo) and placebo (480 mL). Subjects consumed the beverages daily for 4 weeks. Fasting blood samples were analyzed for oxidative stress biomarkers. The intestinal microbiome was analyzed by 16S rRNA sequencing. Compared with the placebo, HCb resulted in a significantly higher increase of total antioxidant capacity (mean ± SD: 1.39 ± 1.69 IU mL-1vs. 0.34 ± 1.73 IU mL-1; p < 0.001) and a higher decrease of the lipid peroxidation product malondialdehyde (-7.29 ± 10.83 nmol mg-1vs. -0.84 ± 15.66 nmol mg-1; p = 0.025). A significant dose-dependent effect on the elevation of superoxide dismutase was observed (p < 0.001). Microbiome data showed that consuming HCb and LCb led to a significant reduction of Pseudomonas (p < 0.05). In conclusion, the current research showed that consuming cranberry beverages significantly improved the antioxidant status in H. pylori positive subjects, which may be related to the reshaping of gut microbiota.

HDAC1 and 2 regulate endothelial VCAM-1 expression and atherogenesis by suppressing methylation of the GATA6 promoter.

Increased expression of vascular cell adhesion molecule (VCAM)-1 on the activated arterial endothelial cell (EC) surface critically contributes to atherosclerosis which may in part be regulated by epigenetic mechanisms. This study investigated whether and how the clinically available histone deacetylases 1 and 2 (HDAC1/2) inhibitor drug Romidepsin epigenetically modulates VCAM-1 expression to suppress atherosclerosis. Methods: VCAM-1 expression was analyzed in primary human aortic EC (HAEC) treated with Romidepsin or transfected with HDAC1/2-targeting siRNA. Methylation of GATA6 promoter region was examined with methylation-specific PCR assay. Enrichment of STAT3 to GATA6 promoter was detected with chromatin immunoprecipitation. Lys685Arg mutation was constructed to block STAT3 acetylation. The potential therapeutic effect of Romidepsin on atherosclerosis was evaluated in Apoe-/- mice fed with a high-fat diet. Results: Romidepsin significantly attenuated TNFα-induced VCAM-1 expression on HAEC surface and monocyte adhesion through simultaneous inhibition of HDAC1/2. This downregulation of VCAM-1 was attributable to reduced expression of transcription factor GATA6. Romidepsin enhanced STAT3 acetylation and its binding to DNA methyltransferase 1 (DNMT1), leading to hypermethylation of the GATA6 promoter CpG-rich region at +140/+255. Blocking STAT3 acetylation at Lys685 disrupted DNMT1-STAT3 interaction, decreased GATA6 promoter methylation, and reversed the suppressive effects of HDAC1/2 inhibition on GATA6 and VCAM-1 expression. Finally, intraperitoneal administration of Romidepsin reduced diet-induced atherosclerotic lesion development in Apoe-/- mice, accompanied by a reduction in GATA6/VCAM-1 expression in the aorta. Conclusions: HDAC1/2 contributes to VCAM-1 expression and atherosclerosis by suppressing STAT3 acetylation-dependent GATA6 promoter methylation. These findings may provide a rationale for HDAC1/2-targeting therapy in atherosclerotic heart disease.

Pathogenesis and Molecular Immune Mechanism of Calcified Aortic Valve Disease.

Calcified aortic valve disease (CAVD) was previously regarded as a passive process associated with valve degeneration and calcium deposition. However, recent studies have shown that the occurrence of CAVD is an active process involving complex changes such as endothelial injury, chronic inflammation, matrix remodeling, and neovascularization. CAVD is the ectopic accumulation of calcium nodules on the surface of the aortic valve, which leads to aortic valve thickening, functional stenosis, and ultimately hemodynamic disorders. CAVD has become an important cause of death from cardiovascular disease. The discovery of therapeutic targets to delay or block the progression of CAVD and the clinical application of transcatheter aortic valve implantation (TAVI) provide new ideas for the prevention and treatment of CAVD. This article summarizes the pathogenesis of CAVD and provides insight into the future directions of CAVD diagnosis and treatment.

IRF-1 mediates the suppressive effects of mTOR inhibition on arterial endothelium.

AIMS: Mammalian target of rapamycin (mTOR) inhibitors used in drug-eluting stents (DES) to control restenosis have been found to delay endothelialization and increase incidence of late-stent thrombosis through mechanisms not completely understood. We revealed that mTOR inhibition (mTORi) upregulated the expression of cell growth suppressor IRF-1 in primary human arterial endothelial cells (HAEC). This study aimed to examine how mTOR-regulated IRF-1 expression contributes to the suppressive effect of mTORi on arterial endothelial proliferation. METHODS AND RESULTS: Western blotting, quantitative PCR, and a dual-luciferase reporter assay indicated that mTOR inhibitors rapamycin and torin 1 upregulated IRF-1 expression and increased its transcriptional activity. IRF-1 in turn contributed to the suppressive effect of mTORi by mediating HAEC apoptosis and cell cycle arrest in part through upregulation of caspase 1 and downregulation of cyclin D3, as revealed by CCK-8 assay, Annexin V binding assay, measurement of activated caspase 3, BrdU incorporation assay, and matrigel tube formation assay. In a mouse model of femoral artery wire injury, administration of rapamycin inhibited EC recovery, an effect alleviated by EC deficiency of IRF-1. Chromatin immunoprecipitation assay with HAEC and rescue expression of wild type or dominant-negative IRF-1 in EC isolated from Irf1-/- mice confirmed transcriptional regulation of IRF-1 on the expression of CASP1 and CCND3. Furthermore, mTORi activated multiple PKC members, among which PKCζ was responsible for the growth-inhibitory effect on HAEC. Activated PKCζ increased IRF1 transcription through JAK/STAT-1 and NF-κB signaling. Finally, overexpression of wild type or mutant raptor incapable of binding mTOR indicated that mTOR-free raptor contributed to PKCζ activation in mTOR-inhibited HAEC. CONCLUSIONS: The study reveals an IRF-1-mediated mechanism that contributes to the suppressive effects of mTORi on HAEC proliferation. Further study may facilitate the development of effective strategies to reduce the side effects of DES used in coronary interventions.

Association of dietary patterns and endoscopic gastric mucosal atrophy in an adult Chinese population.

Atrophy gastritis harbor a high risk for the development of dysplasia and gastric cancer. The study investigated the relationships of specific dietary patterns and endoscopic gastric mucosal atrophy. In this cross-sectional study, we enrolled 574 consecutive outpatients who were diagnosed as chronic gastritis according to endoscopic examination. Dietary intakes of study individuals was assessed using the semi-quantitative food group frequency questionnaire. Logistic regression analyses were used to evaluate the relationship between dietary patterns and endoscopic gastric mucosal atrophy adjusted for potential confounders. A total of 574 participants were included, 286 with endoscopic gastric mucosal atrophy. Three dietary patterns were identified by factor analysis. "Alcohol and fish" (tertile 1 vs. tertile 3: adjusted odds ratio = 1.85, 95% confidence interval: 1.06-3.22) and "coarse cereals" (tertile 1 vs. tertile 3: adjusted odds ratio = 2.05, 95% confidence interval: 1.24-3.39) were associated with an increased risk for endoscopic gastric mucosal atrophy but a "traditional" pattern was not. Dietary pattern was not associated with gastric mucosal atrophy in women or in participants with H. pylori infection. A high adherence to both "Alcohol and Fish" and "Coarse cereals" dietary patterns seem to be associated with higher odds of endoscopic gastric mucosal atrophy in men and in patients without H. pylori infection. Further prospective cohort studies needed to confirm these findings.

The association between functional dyspepsia and depression: a meta-analysis of observational studies.

Concomitant functional dyspepsia (FD) and psychosocial stressors have been reported; however, the association between FD and depression remains controversial and no quantitative meta-analysis exists. Published articles were identified through a comprehensive review of PubMed, Embase, and Web of Science from inception to the 8 July 2018. The pooled odds ratios (ORs) with 95% confidence intervals and subgroup analyses were calculated using a random-effects model. Findings for a total of 59 029 individuals were pooled across 23 studies and examined. Our analyses showed a positive association between FD and depression, with an OR of 2.28 (95% confidence interval: 2.02-3.81; I=100%). In the subgroup analysis, FD patients in Europe (OR=6.19) were more likely to have depression compared with Asians (OR=2.47); the overall significance results decreased the most in subgroup which the overall significance of the subgroup analyses results decreased the most in studies that adjusted for BMI (OR=1.42). Our meta-analysis showed a positive association between FD and depression. Further large-scale prospective cohort studies are needed to investigate the causality between FD and depression.

Downregulation of GATA6 in mTOR-inhibited human aortic endothelial cells: effects on TNF-α-induced VCAM-1 expression and monocytic cell adhesion.

Increased expression of vascular cell adhesion molecule 1 (VCAM-1) on the aortic endothelium is an early marker of atherogenesis, promoted in part by elevated levels of inflammatory cytokines such as TNF-α. Mammalian target of rapamycin (mTOR) is a ubiquitous signaling molecule that has been considered to contribute to diverse cellular processes through mTOR complex 1 (mTORC1) or complex 2 (mTORC2). This study aimed to elucidate the role of mTOR signaling in TNF-α-induced VCAM-1 expression by the arterial endothelium. Primary human aortic endothelial cells (HAECs) were treated with low-dose (0.1 ng/ml) TNF-α, and VCAM-1 expression was measured by real-time quantitative PCR, Western blot analysis, and flow cytometry. Inhibition of mTOR through siRNA-mediated depletion or treatment with chemical inhibitors rapamycin or torin 1 suppressed VCAM1 transcription, which translated to inhibition of VCAM-1 surface expression by HAECs and concomitant decreased adhesion of monocytes. A promoter luciferase assay and chromatin immunoprecipitation indicated that mTOR regulated VCAM1 transcription through a mechanism involving transcription factor GATA6. Activation of PKC-α and an increase in miR-200a-3p expression, caused by mTOR inhibition but not disruption of mTORC1 or mTORC2 singly or together, decreased TNF-α-induced GATA6 expression and its enrichment at the VCAM1 promoter. In conclusion, mTOR inhibition activates PKC-α independently of disruption of mTORC1 and/or mTORC2, which challenges the conventional wisdom regarding mTOR signaling. Moreover, mTOR signals through transcriptional and posttranscriptional mechanisms to elicit maximal cytokine-induced endothelial inflammation that precedes atherosclerosis. NEW & NOTEWORTHY Both mammalian target of rapamycin (mTOR) complex 1 (mTORC1) and mTORC2 contribute to PKC-α activation in the human aortic endothelium. Inhibition of mTOR is not equivalent to disruption of mTORC1 and/or mTORC2 in affecting human aortic endothelial cell signaling. Specifically, inhibition of mTOR causes PKC-α activation and miR-200a-3p upregulation, which independently suppresses TNF-α-induced transcription factor GATA6 expression and subsequently inhibits VCAM-1 expression and monocytic cell adhesion onto the aortic endothelium.

11,12-Epoxyecosatrienoic acids mitigate endothelial dysfunction associated with estrogen loss and aging: Role of membrane depolarization.

OBJECTIVE: Endothelial dysfunction, including upregulation of inflammatory adhesion molecules and impaired vasodilatation, is a key element in cardiovascular disease. Aging and estrogen withdrawal in women are associated with endothelial inflammation, vascular stiffness and increased cardiovascular disease. Epoxyecosatrienoic acids (EETs), the products of arachidonic acid metabolism mediated by cytochrome P450 (CYP) 2J, 2C and other isoforms, are regulated by soluble epoxide hydrolase (sEH)-catalyzed conversion into less active diols. We hypothesized that 11,12-EETs would reduce the endothelial dysfunction associated with aging and estrogen loss. APPROACH/RESULTS: When stabilized by an sEH inhibitor (seHi), 11,12-EET at a physiologically low dose (0.1nM) reduced cytokine-stimulated upregulation of adhesion molecules on human aorta endothelial cells (HAEC) and monocyte adhesion under shear flow through marked depolarization of the HAEC when combined with TNFα. Mechanistically, neither 11,12-EETs nor 17ß-estradiol (E2) at physiologic concentrations prevented activation of NFκB by TNFα. E2 at physiological concentrations reduced sEH expression in HAEC, but did not alter CYP expression, and when combined with TNFα depolarized the cell. We also examined vascular dysfunction in adult and aged ovariectomized Norway brown rats (with and without E2 replacement) using an ex-vivo model to analyze endothelial function in an intact segment of artery. sEHi and 11,12-EET with or without E2 attenuated phenylephrine induced constriction and increased endothelial-dependent dilation of aortic rings from ovariectomized rats. CONCLUSIONS: Increasing 11,12-EETs through sEH inhibition effectively attenuates inflammation and may provide an effective strategy to preserve endothelial function and prevent atherosclerotic heart disease in postmenopausal women.

Interleukin 18 function in atherosclerosis is mediated by the interleukin 18 receptor and the Na-Cl co-transporter.

Interleukin-18 (IL18) participates in atherogenesis through several putative mechanisms. Interruption of IL18 action reduces atherosclerosis in mice. Here, we show that absence of the IL18 receptor (IL18r) does not affect atherosclerosis in apolipoprotein E-deficient (Apoe(-/-)) mice, nor does it affect IL18 cell surface binding to or signaling in endothelial cells. As identified initially by co-immunoprecipitation with IL18, we found that IL18 interacts with the Na-Cl co-transporter (NCC; also known as SLC12A3), a 12-transmembrane-domain ion transporter protein preferentially expressed in the kidney. NCC is expressed in atherosclerotic lesions, where it colocalizes with IL18r. In Apoe(-/-) mice, combined deficiency of IL18r and NCC, but not single deficiency of either protein, protects mice from atherosclerosis. Peritoneal macrophages from Apoe(-/-) mice or from Apoe(-/-) mice lacking IL18r or NCC show IL18 binding and induction of cell signaling and cytokine and chemokine expression, but macrophages from Apoe(-/-) mice with combined deficiency of IL18r and NCC have a blunted response. An interaction between NCC and IL18r on macrophages was detected by co-immunoprecipitation. IL18 binds to the cell surface of NCC-transfected COS-7 cells, which do not express IL18r, and induces cell signaling and cytokine expression. This study identifies NCC as an IL18-binding protein that collaborates with IL18r in cell signaling, inflammatory molecule expression, and experimental atherogenesis.

Triglyceride-rich lipoprotein modulates endothelial vascular cell adhesion molecule (VCAM)-1 expression via differential regulation of endoplasmic reticulum stress.

Circulating triglyceride-rich lipoproteins (TGRL) from hypertriglyceridemic subjects exacerbate endothelial inflammation and promote monocyte infiltration into the arterial wall. We have recently reported that TGRL isolated from human blood after a high-fat meal can elicit a pro- or anti-atherogenic state in human aortic endothelial cells (HAEC), defined as up- or down-regulation of VCAM-1 expression in response to tumor necrosis factor alpha (TNFα) stimulation, respectively. A direct correlation was found between subjects categorized at higher risk for cardiovascular disease based upon serum triglycerides and postprandial production of TGRL particles that increased VCAM-1-dependent monocyte adhesion to inflamed endothelium. To establish how TGRL metabolism is linked to VCAM-1 regulation, we examined endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) pathways. Regardless of its atherogenicity, the rate and extent of TGRL internalization and lipid droplet formation by HAEC were uniform. However, pro-atherogenic TGRL exacerbated ER membrane expansion and stress following TNFα stimulation, whereas anti-atherogenic TGRL ameliorated such effects. Inhibition of ER stress with a chemical chaperone 4-phenylbutyric acid decreased TNFα-induced VCAM-1 expression and abrogated TGRL's atherogenic effect. Activation of ER stress sensors PKR-like ER-regulated kinase (PERK) and inositol requiring protein 1α (IRE1α), and downstream effectors including eukaryotic initiation factor-2α (eIF2α), spliced X-box-binding protein 1 (sXBP1) and C/EBP homologous protein (CHOP), directly correlated with the atherogenic activity of an individual's TGRL. Modulation of ER stress sensors also correlated with changes in expression of interferon regulatory factor 1 (IRF-1), a transcription factor of Vcam-1 responsible for regulation of its expression. Moreover, knockdown studies using siRNA defined a causal relationship between the PERK/eIF2α/CHOP pathway and IRF-1-mediated VCAM-1 expression. We conclude that ER stress and the UPR contribute to the regulation of Vcam-1 transcription as a function of the atherogenic nature of TGRL.

Shear stress modulates VCAM-1 expression in response to TNF-α and dietary lipids via interferon regulatory factor-1 in cultured endothelium.

Dyslipidemia is a primary risk factor for cardiovascular disease, but the specific mechanisms that determine the localization of atherosclerotic plaques in arteries are not well defined. Triglyceride-rich lipoproteins (TGRL) isolated from human plasma after a high-fat meal modulate TNF-α-induced VCAM-1 expression in cultured human aortic endothelial cells (HAECs) via an interferon regulatory factor (IRF)-1-dependent transcriptional mechanism. We examined whether fluid shear stress acts as a mediator of IRF-1-dependent VCAM-1 expression in response to cytokine and dietary lipids. IRF-1 and VCAM-1 were examined by immunofluorescence in TNF-α-stimulated HAEC monolayers exposed to TGRL and a linear gradient of shear stress ranging from 0 to 16 dyn/cm(2) in a microfluidic device. Shear stress alone modulated TNF-α-induced VCAM-1 expression, eliciting a 150% increase at low shear stress (2 dyn/cm(2)) and a 70% decrease at high shear stress (12 dyn/cm(2)) relative to static. These differences correlated with a 60% increase in IRF-1 expression under low shear stress and a 40% decrease under high shear stress. The addition of TGRL along with cytokine activated a fourfold increase in VCAM-1 expression and a twofold increase in IRF-1 expression. The combined effect of shear stress and TGRL on the upregulation of membrane VCAM-1 was abolished by transfection of HAECs with IRF-1-specific small interfering RNA. In a healthy swine model, elevated levels of endothelial IRF-1 were also observed within atherosusceptible regions of the aorta by Western blot analysis and immunohistochemistry, implicating arterial hemodynamics in the regulation of IRF-1 expression. These data demonstrate direct roles for fluid shear stress and postprandial TGRL from human serum in the regulation of IRF-1 expression and downstream inflammatory responses in HAECs.

ADAM15 deficiency attenuates pulmonary hyperpermeability and acute lung injury in lipopolysaccharide-treated mice.

ADAM15 is a disintegrin and metalloprotease recently implicated in cancer and chronic immune disorders. We have recently characterized ADAM15 as a mediator of endothelial barrier dysfunction. Whether this molecule contributes to acute inflammation has not been evaluated. The purpose of this study was to investigate the role of ADAM15 in mediating pulmonary microvascular leakage during acute inflammatory injury. Immunofluorescent staining and Western blotting revealed that the endothelium was the main source of ADAM15 in lung tissue. In a mouse model of acute lung injury induced by lipopolysaccharide (LPS), upregulation of ADAM15 was observed in association with pulmonary edema and neutrophil infiltration. The LPS-induced inflammatory injury, as demonstrated by bronchoalveolar lavage neutrophil count, lung wet-to-dry weight ratio, and myeloperoxidase activity, was significantly attenuated in Adam15(-/-) mice. Studies with primary cell culture demonstrated abundant ADAM15 expression in endothelial cells (ECs) of mouse lung but not in neutrophils. Deficiency of ADAM15 in ECs had no obvious effect on basal permeability but significantly attenuated hyperpermeability response to LPS as evidenced by albumin flux assay and measurements of transendothelial electrical resistance, respectively. ADAM15 deficiency also reduced neutrophil chemotactic transmigration across endothelial barriers in the presence or absence of formyl-methionyl-leucyl-phenylalanine (fMLP). Rescue expression of ADAM15 in Adam15(-/-) ECs restored neutrophil transendothelial migration. These data indicate that ADAM15 upregulation contributes to inflammatory lung injury by promoting endothelial hyperpermeability and neutrophil transmigration.

IRF-1 and miRNA126 modulate VCAM-1 expression in response to a high-fat meal.

RATIONALE: A high-fat diet accompanied by hypertriglyceridemia increases an individual's risk for development of atherosclerosis. An early event in this process is monocyte recruitment through binding to vascular cell adhesion molecule 1 (VCAM-1) upregulated on inflamed arterial endothelium. Diets high in polyunsaturated fatty acids (PUFAs) may provide athero-protection by ameliorating this effect. OBJECTIVE: We investigated the acute regulation of VCAM-1 expression in human aortic endothelial cells (HAEC) in response to triglyceride-rich lipoproteins (TGRL) isolated from subjects after consumption of a high-fat meal. METHODS AND RESULTS: Postprandial TGRL isolated from 38 subjects were categorized as proatherogenic or antiatherogenic according to their capacity to alter the inflammatory response of HAEC. Proatherogenic TGRL increased expression of VCAM-1, intercellular adhesion molecule 1 (ICAM-1), and E-selectin by ≈20% compared with stimulation with tumor necrosis factor-α alone, whereas antiatherogenic TGRL decreased VCAM-1 expression by ≈20% while still upregulating ICAM-1. The relative atherogenicity of TGRL positively correlated with particle density of TG, apolipoprotein (Apo)CIII, ApoE, and cholesterol. Ω3-PUFA mimicked the effect of antiatherogenic TGRL by downregulating VCAM-1 expression. TGRL exerted this differential regulation of VCAM-1 by reciprocally modulating expression and activity of the transcription factor interferon regulatory factor 1 (IRF-1) and expression of microRNA 126 (miR-126). Overexpression or silencing of IRF-1 or miR-126 expression recapitulated the proatherogenic or antiatherogenic regulation of VCAM-1. CONCLUSIONS: In response to a high-fat meal, TGRL bias the inflammatory response of endothelium via transcriptional and posttranscriptional editing of VCAM-1. Subjects with an anti-inflammatory response to a meal produced TGRL that was enriched in nonesterified fatty acids, decreased IRF-1 expression, increased miR-126 activity, and diminished monocyte arrest.

A disintegrin and metalloproteinase 15 contributes to atherosclerosis by mediating endothelial barrier dysfunction via Src family kinase activity.

OBJECTIVE: Endothelium dysfunction is an initiating factor in atherosclerosis. A disintegrin and metalloproteinase 15 (ADAM 15) is a multidomain metalloprotease recently identified as a regulator of endothelial permeability. However, whether and how ADAM15 contributes to atherosclerosis remains unknown. METHODS AND RESULTS: Genetic ablation of ADAM15 in apolipoprotein E-deficient mice led to a significant reduction in aortic atherosclerotic lesion size (by 52%), plaque macrophage infiltration (by 69%), and smooth muscle cell deposition (by 82%). In vitro studies implicated endothelial-derived ADAM15 in barrier dysfunction and monocyte transmigration across mouse aortic and human umbilical vein endothelial cell monolayers. This role of ADAM15 depended on intact functioning of the cytoplasmic domain, as evidenced in experiments with site-directed mutagenesis targeting the metalloprotease active site (E349A), the disintegrin domain (Arginine-Glycine-Aspartic acid→Threonine-Aspartic acid-Aspartic acid), or the cytoplasmic tail. Further investigations revealed that ADAM15-induced barrier dysfunction was concomitant with dissociation of endothelial adherens junctions (vascular endothelial [VE]-cadherin/γ-catenin), an effect that was sensitive to Src family kinase inhibition. Through small interfering RNA-mediated knockdown of distinct Src family kinase members, c-Src and c-Yes were identified as important mediators of these junctional effects of ADAM15. CONCLUSIONS: These results suggest that endothelial cell-derived ADAM15, signaling through c-Src and c-Yes, contributes to atherosclerotic lesion development by disrupting adherens junction integrity and promoting monocyte transmigration.

Role of non-muscle myosin light chain kinase in neutrophil-mediated intestinal barrier dysfunction during thermal injury.

Neutrophils and non-muscle myosin light chain kinase (nmMLCK) have been implicated in intestinal microvascular leakage and mucosal hyperpermeability in inflammation and trauma. The aim of this study was to characterize the role of nmMLCK in neutrophil-dependent gut barrier dysfunction following thermal injury, a common form of trauma that typically induces inflammation in multiple organs. Histopathological examination of the small intestine in mice after a full-thickness burn revealed morphological evidence of mucosa inflammation characterized by neutrophil infiltration into the lamina propria, epithelial contraction, and narrow villi with blunt brush borders and loss of goblet cells. Compared with their wild-type counterparts, nmMLCK mice displayed diminished morphological abnormalities. Likewise, intravital microscopic studies showed significant leukocyte adhesion in intestinal microvessels after burn, a response that was blunted in the absence of nmMLCK. Functionally, thermal injury significantly increased the gut lumen-to-blood transport of fluorescein isothiocyanate-dextran (4 kd), and this hyperpermeability was attenuated by either neutrophil depletion or nmMLCK deficiency. Consistent with the in vivo observations, in vitro assays with Caco-2 epithelial cell monolayers revealed a decrease in transcellular electric resistance coupled with myosin light chain phosphorylation, actomyosin ring condensation, and claudin-1 internalization upon stimulation with fMLP (N-formyl-methionyl-leucyl-phenylalanine)-activated neutrophils. Pretreatment of the cells with the MLCK inhibitor ML-7 prevented the tight junction responses. Taken together, the results suggest that nmMLCK plays an important role in neutrophil-dependent intestinal barrier dysfunction during inflammatory injury.

Nonmuscle myosin light-chain kinase deficiency attenuates atherosclerosis in apolipoprotein E-deficient mice via reduced endothelial barrier dysfunction and monocyte migration.

BACKGROUND: Endothelial dysfunction and monocyte migration are key events in the pathogenesis of atherosclerosis. Nonmuscle myosin light-chain kinase (nmMLCK), the predominant MLCK isoform in endothelial cells, has been shown to contribute to vascular inflammation by altering endothelial barrier function. However, its impact on atherogenesis remains unknown. METHODS AND RESULTS: We investigated the role of nmMLCK in the development of atherosclerotic lesions in apolipoprotein E-deficient (apoE(-/-)) mice fed an atherogenic diet for 12 weeks. Histopathological examination demonstrated that nmMLCK deficiency (apoE(-/-)nmmlck(-/-)) reduced the size of aortic lesions by 53%, lipid contents by 44%, and macrophage deposition by 40%. Western blotting and reverse-transcription polymerase chain reaction revealed the expression of nmMLCK in aortic endothelial cells and peripheral blood monocytes. Measurements of transendothelial electric resistance indicated that nmMLCK deficiency attenuated endothelial barrier dysfunction caused by thrombin, oxidized low-density lipoprotein, and tumor necrosis factor α. In monocytes, nmMLCK deficiency reduced their migration in response to the chemokine monocyte chemoattractant protein-1. Further mechanistic studies showed that nmMLCK acted through both myosin light chain phosphorylation-coupled and -uncoupled pathways; the latter involved Rous sacracoma virus homolog genes-encoded tyrosine kinases (Src) signaling. Moreover, depletion of Src via gene silencing, site-specific mutagenesis, or pharmacological inhibition of Src greatly attenuated nmMLCK-dependent endothelial barrier dysfunction and monocyte migration. CONCLUSIONS: Nonmuscle myosin light-chain kinase contributes to atherosclerosis by regulating endothelial barrier function and monocyte migration via mechanisms involving not only kinase-mediated MLC phosphorylation but also Src activation.