Atherosclerosis is a chronic inflammatory disease of the vessel wall. Recent evidence suggests that chronic vascular inflammation ensues as an imbalance between pro-and anti-inflammatory mediators. Recently identified lipid mediators (eg, lipoxins and resolvins) play active roles in promoting the resolution of inflammation. Alterations in vascular smooth muscle cell (VSMC) phenotype, which manifest as a loss of contractile protein expression and increased proliferation and migration, are prominent mechanistic features of both atherosclerosis and restenosis following various interventions (eg, angioplasty and bypass grafting). We sought to determine whether human atherosclerosis is associated with a "resolution deficit" and whether lipoxins and resolvins influence VSMC phenotype. Here we report that plasma levels of aspirin-triggered lipoxin are significantly lower in patients with symptomatic peripheral artery disease than in healthy volunteers. Both aspirintriggered lipoxin and resolvin E1 block platelet-derived growth factor-stimulated migration of human saphenous vein SMCs and decrease phosphorylation of the platelet-derived growth factor receptor-. Importantly, receptors for aspirin-triggered lipoxin and resolvin E1 (ALX and ChemR23, respectively) were identified in human VSMCs. Overall, these results demonstrate that stimulatory lipid mediators confer a protective phenotypic switch in VSMCs and elucidate new functions for these mediators in the regulation of SMC biology. These results also suggest that peripheral artery disease is associated with an inflammation-resolution deficit and highlight a potential therapeutic opportunity for the regulation of vascular injury responses. (Am J Pathol
Genetic variation at the chromosome 9p21 risk locus promotes cardiovascular disease; however, it is unclear how or which proteins encoded at this locus contribute to disease. We have previously demonstrated that loss of one candidate gene at this locus, cyclin-dependent kinase inhibitor 2B (Cdkn2b), in mice promotes vascular SMC apoptosis and aneurysm progression. Here, we investigated the role of Cdnk2b in atherogenesis and found that in a mouse model of atherosclerosis, deletion of Cdnk2b promoted advanced development of atherosclerotic plaques composed of large necrotic cores. Furthermore, human carriers of the 9p21 risk allele had reduced expression of CDKN2B in atherosclerotic plaques, which was associated with impaired expression of calreticulin, a ligand required for activation of engulfment receptors on phagocytic cells. As a result of decreased calreticulin, CDKN2B-deficient apoptotic bodies were resistant to efferocytosis and not efficiently cleared by neighboring macrophages. These uncleared SMCs elicited a series of proatherogenic juxtacrine responses associated with increased foam cell formation and inflammatory cytokine elaboration. The addition of exogenous calreticulin reversed defects associated with loss of Cdkn2b and normalized engulfment of Cdkn2b-deficient cells. Together, these data suggest that loss of CDKN2B promotes atherosclerosis by increasing the size and complexity of the lipid-laden necrotic core through impaired efferocytosis.
Fibronectin (fn) is an extracellular matrix (ECM) molecule important in cell adhesion and migration and in wound healing. It is also likely important in periodontal ligament (PDL) cell-ECM interactions, and thus in regenerating periodontal tissues. In this study we characterized PDL cells and their interactions with FN, testing different PDL cell isolates taken from healthy and diseased conditions. PDL cells were characterized by their morphology, integrin profile, motility, and bone nodule formation. Cells were then assayed for adhesion, proliferation, and chemotaxis in response to FN or FN fragments. Cell isolates were morphologically heterogeneous and fibroblastic, had a normal-appearing actin cytoskeleton and a wide range of migration potentials, and formed bone-like nodules in vitro. They expressed alpha5, beta1, alpha v, and alpha4 integrin subunits, known receptors for FN, and in fact they bound FN preferentially at 5 and 10 microg/ml. Intact FN induced greater PDL cell proliferation and chemotaxis than did FN fragments (120-kDa cell-binding, 60-kDa heparin-binding, and 45-kDa collagen-binding). PDL cells harvested from diseased and healthy conditions were no different on the basis of these assays. These data demonstrate that PDL cells are a mixed population of fibroblastic cells, capable of forming a mineralized matrix. They also suggest that maximal proliferation and chemotaxis require specific FN domains that are present on the intact molecule but not its fragments.
Myxococcus xanthus is a gliding bacterium that possesses two motility systems, the adventurous (A-motility) and social (S-motility) systems. A-motility is used for individual cell gliding, while S-motility is used for gliding in multicellular groups. Video microscopy studies showed that nla24 cells are non-motile on agar surfaces, suggesting that the nla24 gene product is absolutely required for both A-motility and S-motility under these assay conditions. S-motility requires functional type IV pili, wild-type LPS O-antigen, and an extracellular matrix of exopolysaccharide (EPS) and protein called fibrils. The results of expression studies and tethering assays indicate that the nla24 mutant has functional type IV pili. The nla24 mutant also produces wild-type LPS. However, several lines of evidence suggest that the nla24 mutant is defective for production of the EPS portion of the fibril matrix. The nla24 mutant is also defective for transcription of two genes (aglU and cglB) known to be required for A-motility, which is consistent with the idea that nla24 cells are defective for A-motility. Based on these findings, it is proposed that the putative transcriptional activator Nla24 regulates a subset of genes that are important for A-motility and S-motility in M. xanthus.
Single-cell technologies have immense potential to shed light on molecular and biological processes that drive human diseases. Mass cytometry (or Cytometry by Time Of Flight mass spectrometry, CyTOF) has already been employed in clinical studies to comprehensively survey patients’ circulating immune system. As interest in the “bedside” application of mass cytometry is growing, the delineation of relevant methodological issues is called for. This report uses a newly generated dataset to discuss important methodological considerations when mass cytometry is implemented in a clinical study. Specifically, the use of whole blood samples versus peripheral blood mononuclear cells (PBMCs), design of mass-tagged antibody panels, technical and analytical implications of sample barcoding, and application of traditional and unsupervised approaches to analyze high-dimensional mass cytometry datasets are discussed. A mass cytometry assay was implemented in a cross-sectional study of 19 women with a history of term or preterm birth to determine whether immune traits in peripheral blood differentiate the two groups in the absence of pregnancy. Twenty-seven phenotypic and 11 intracellular markers were simultaneously analyzed in whole blood samples stimulated with lipopolysaccharide (LPS at 0, 0.1, 1, 10, and 100 ng mL−1) to examine dose-dependent signaling responses within the toll-like receptor 4 (TLR4) pathway. Complementary analyses, grounded in traditional or unsupervised gating strategies of immune cell subsets, indicated that the prpS6 and pMAPKAPK2 responses in classical monocytes are accentuated in women with a history of preterm birth (FDR<1%). The results suggest that women predisposed to preterm birth may be prone to mount an exacerbated TLR4 response during the course of pregnancy. This important hypothesis-generating finding points to the power of single-cell mass cytometry to detect biologically important differences in a relatively small patient cohort.
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