Elevated triglyceride-rich lipoproteins (TGRL) in circulation is a risk factor for atherosclerosis. TGRL from subjects consuming a high saturated fat test meal elicited a variable inflammatory ...response in TNFα-stimulated endothelial cells (EC) that correlated strongly with the polyunsaturated fatty acid (PUFA) content. This study investigates how the relative abundance of oxygenated metabolites of PUFA, oxylipins, is altered in TGRL postprandially, and how these changes promote endothelial inflammation. Human aortic EC were stimulated with TNFα and treated with TGRL, isolated from subjects' plasma at fasting and 3.5 hrs postprandial to a test meal high in saturated fat. Endothelial VCAM-1 surface expression stimulated by TNFα provided a readout for atherogenic inflammation. Concentrations of esterified and non-esterified fatty acids and oxylipins in TGRL were quantified by mass spectrometry. Dyslipidemic subjects produced TGRL that increased endothelial VCAM-1 expression by ≥35%, and exhibited impaired fasting lipogenesis activity and a shift in soluble epoxide hydrolase and lipoxygenase activity. Pro-atherogenic TGRL were enriched in eicosapentaenoic acid metabolites and depleted in esterified C18-PUFA-derived diols. Abundance of these metabolites was strongly predictive of VCAM-1 expression. We conclude the altered metabolism in dyslipidemic subjects produces TGRL with a unique oxylipin signature that promotes a pro-atherogenic endothelial phenotype.
Neutrophils are essential to protect the host against invading pathogens but can promote disease progression in sickle cell disease (SCD) by becoming adherent to inflamed microvascular networks in ...peripheral tissue throughout the body. During the inflammatory response, leukocytes extravasate from the bloodstream using selectin adhesion molecules and migrate to sites of tissue insult through activation of integrins that are essential for combating pathogens. However, during vaso-occlusion associated with SCD, neutrophils are activated during tethering and rolling on selectins upregulated on activated endothelium that line blood vessels. Recently, we reported that recognition of sLe
on L-selectin by E-selectin during neutrophil rolling initiates shear force resistant catch-bonds that facilitate tethering to endothelium and activation of integrin bond clusters that anchor cells to the vessel wall. Evidence indicates that blocking this important signaling cascade prevents the congestion and ischemia in microvasculature that occurs from neutrophil capture of sickled red blood cells, which are normally deformable ellipses that flow easily through small blood vessels. Two recently completed clinical trials of therapies targeting selectins and their effect on neutrophil activation in small blood vessels reveal the importance of mechanoregulation that in health is an immune adaption facilitating rapid and proportional leukocyte adhesion, while sustaining tissue perfusion. We provide a timely perspective on the mechanism underlying vaso-occlusive crisis (VOC) with a focus on new drugs that target selectin mediated integrin adhesive bond formation.
T cell cytokines contribute to immunity against Staphylococcus aureus, but the predominant T cell subsets involved are unclear. In an S. aureus skin infection mouse model, we found that the IL- 17 ...response was mediated by γδ T cells, which trafficked from lymph nodes to the infected skin to induce neutrophil recruitment, proinflammatory cytokines IL-1α, IL-1β, and TNF, and host defense peptides. RNA-seq for TRG and TRD sequences in lymph nodes and skin revealed a single clonotypic expansion of the encoded complementarity-determining region 3 amino acid sequence, which could be generated by canonical nucleotide sequences of TRGV5 or TRGV6 and TRDV4. However, only TRGV6 and TRDV4 but not TRGV5 sequences expanded. Finally, Vγ6⁺ T cells were a predominant γδ T cell subset that produced IL-17A as well as IL-22, TNF, and IFNγ, indicating a broad and substantial role for clonal Vγ6⁺Vδ4⁺ T cells in immunity against S. aureus skin infections.
Discovery of new genes and proteins directly supporting leukocyte adhesion is waning, whereas there is heightened interest in the cell mechanics and receptor dynamics that lead from transient ...tethering via selectins to affinity shifts and adhesion strengthening through integrins. New optical tools enable real-time imaging of leukocyte rolling and arrest in parallel plate flow channels (PPFCs), and detection of single-molecule force spectroscopy provides an inner view of the intercellular adhesive contact region. Leukocyte recruitment during acute inflammation is triggered by ligation of G protein-coupled chemotactic receptors (GPCRs) and clustering of selectins. This, in turn, activates beta(2)-integrin (CD18), which facilitates cell capture and arrest in shear flow. This review provides a conceptual model for the molecular events supporting leukocyte recruitment.
Recognition of integrins by CD62P has not been reported and this motivated a docking simulation using integrin αvβ3 as a target. We predicted that the C-type lectin domain of CD62P functions as a ...potential integrin ligand and observed that it specifically bound to soluble β3 and β1 integrins. Known inhibitors of the interaction between CD62P-PSGL-1 did not suppress the binding, whereas the disintegrin domain of ADAM-15, a known integrin ligand, suppressed recognition by the lectin domain. Furthermore, an R16E/K17E mutation in the predicted integrin-binding interface located outside of the glycan-binding site within the lectin domain, strongly inhibited CD62P binding to integrins. In contrast, the E88D mutation that strongly disrupts glycan binding only slightly affected CD62P-integrin recognition, indicating that the glycan and integrin-binding sites are distinct. Notably, the lectin domain allosterically activated integrins by binding to the allosteric site 2. We conclude that CD62P-integrin binding may function to promote a diverse set of cell-cell adhesive interactions given that β3 and β1 integrins are more widely expressed than PSGL-1 that is limited to leukocytes.
Endothelial up‐regulation of VCAM‐1 at susceptible sites in arteries modulates the recruitment efficiency of inflammatory monocytes that initiates atherosclerotic lesion formation. We reported that ...hydrodynamic shear stress (SS) mechanoregulates inflammation in human aortic endothelial cells through endoplasmic reticulum (ER) stress via activation of the transcription factor x‐box binding protein 1 (XBP1). Here, a microfluidic flow channel that produces a linear gradient of SS along a continuous monolayer of endothelium was used to delve the mechanisms underlying transcriptional regulation of TNF‐α‐stimulated VCAM‐1 expression. High‐resolution immunofluorescence imaging enabled continuous detection of platelet endothelial cell adhesion molecule 1 (PECAM‐1)‐dependent, outside‐in signaling as a function of SS magnitude. Differential expression of VCAM‐1 and intercellular adhesion molecule 1 (ICAM‐1) was regulated by the spatiotemporal activation of MAPKs, ER stress markers, and transcription factors, which was dependent on the mechanosensing of SS through PECAM‐1 and PI3K. Inhibition of p38 specifically abrogated the rise to peak VCAM‐1 at low SS (2 dyn/cm2), whereas inhibition of ERK1/2 attenuated peak ICAM‐1 at high SS (12 dyn/cm2). A shear stress‐regulated temporal rise in p38 phosphorylation activated the nuclear translocation of XBP1, which together with the transcription factor IFN regulatory factor 1, promoted maximum VCAM‐1 expression. These data reveal a mechanism by which SS sensitizes the endothelium to a cytokine‐induced ER stress response to spatially regulate inflammation promoting atherosclerosis.—Bailey, K. A., Moreno, E., Haj, F. G., Simon, S. I., Passerini, A. G. Mechanoregulation of p38 activity enhances endoplasmic reticulum stress‐mediated inflammation by arterial endothelium. FASEB J. 33, 12888–12899 (2019). www.fasebj.org
Orai1 was reported to function as a calcium channel subunit that facilitates store operated calcium entry (SOCE) in T cells and is necessary for formation of the immune synapse. We reasoned that SOCE ...via Orai1 might regulate PMNs activation during recruitment to inflamed endothelium. Orai1 function was assessed by real-time imaging of calcium transients as PMNs were stimulated to roll, arrest, and migrate on E-selectin and ICAM-1 in shear flow. Calcium entry was significantly reduced when Orai1 function was impaired by heterozygous knockout in a mouse model or by siRNA knockdown in HL-60 cells. Reduced Orai-1 expression correlated with the delayed onset of arrest and reduced ability to transition to a polarized migratory phenotype. Inhibition of SOCE by treatment with 2-APB, or blocking phospholipase C (PLC) mediated calcium store release with U73122, abrogated formyl peptide induced calcium elevation, and delayed subsequent cell arrest and polarization. These results suggest that calcium entry via Orai1 is the predominant SOCE that cooperates with cytoplasmic calcium store release in coordinating integrin-dependent PMN arrest and migration in the acute response to inflammation.
Acute inflammation triggers the innate immune response of neutrophils that efficiently traffic from the bloodstream to concentrate at high numbers at the site of tissue infection or wounding. A ...gatekeeper in this process is activation of β(2) integrins, which form bond clusters with ICAM-1 on the endothelial surface. These bond clusters serve dual functions of providing adhesive strength to anchor neutrophils under the shear forces of blood flow and directional guidance for cell polarization and subsequent transmigration on inflamed endothelium. We hypothesized that shear forces transmitted through high-affinity LFA-1 facilitates the cooperation with the calcium release-activated channel Orai1 in directing localized cytoskeletal activation and directed migration. By using vascular mimetic microfluidic channels, we observed neutrophil arrest on a substrate of either ICAM-1 or allosteric Abs that stabilize a high- or low-affinity conformation of LFA-1. Neutrophils captured via low-affinity LFA-1 did not exhibit intracellular calcium flux, F-actin polymerization, cell polarization, or directional migration under shear flow. In contrast, high-affinity LFA-1 provided orientation along a uropod-pseudopod axis that required calcium flux through Orai1. We demonstrate how the shear stress of blood flow can transduce distinct outside-in signals at focal sites of high-affinity LFA-1 that provide contact-mediated guidance for neutrophil emigration.
To examine infiltration of blood foamy monocytes, containing intracellular lipid droplets, into early atherosclerotic lesions and its contribution to development of nascent atherosclerosis.
In ...apoE(-/-) mice fed Western high-fat diet (WD), >10% of circulating monocytes became foamy monocytes at 3 days on WD and >20% of monocytes at 1 week. Foamy monocytes also formed early in blood of Ldlr(-/-)Apobec1(-/-) (LDb) mice on WD. Based on CD11c and CD36, mouse monocytes were categorized as CD11c(-)CD36(-), CD11c(-)CD36(+), and CD11c(+)CD36(+). The majority of foamy monocytes were CD11c(+)CD36(+), whereas most nonfoamy monocytes were CD11c(-)CD36(-) or CD11c(-)CD36(+) in apoE(-/-) mice on WD. In wild-type mice, CD11c(+)CD36(+) and CD11c(-)CD36(+), but few CD11c(-)CD36(-), monocytes took up cholesteryl ester-rich very low-density lipoproteins (CE-VLDLs) isolated from apoE(-/-) mice on WD, and CE-VLDL uptake accelerated CD11c(-)CD36(+) to CD11c(+)CD36(+) monocyte differentiation. Ablation of CD36 decreased monocyte uptake of CE-VLDLs. Intravenous injection of DiI-CE-VLDLs in apoE(-/-) mice on WD specifically labeled CD11c(+)CD36(+) foamy monocytes, which infiltrated into nascent atherosclerotic lesions and became CD11c(+) cells that were selectively localized in atherosclerotic lesions. CD11c deficiency reduced foamy monocyte infiltration into atherosclerotic lesions. Specific and consistent depletion of foamy monocytes (for 3 weeks) by daily intravenous injections of low-dose clodrosome reduced development of nascent atherosclerosis.
Foamy monocytes, which form early in blood of mice with hypercholesterolemia, infiltrate into early atherosclerotic lesions in a CD11c-dependent manner and play crucial roles in nascent atherosclerosis development.
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.
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.
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.
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•Raptor contributed to PKCζ activation in mTOR-inhibited aortic EC independent of mTORC1.•PKCζ activated the JAK/STAT-1 and NF-κB signaling pathways, resulting in upregulation of IRF-1.•IRF-1 mediated the suppressive effects of mTOR inhibition on EC through transcriptional regulation of CASP1 and CCND3.•EC-specific deletion of IRF-1 was associated with enhanced EC recovery and reduced intimal hyperplasia in vivo.
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