LXR signaling pathways link cholesterol metabolism with risk for prediabetes and diabetes.

BACKGROUNDPreclinical studies suggest that cholesterol accumulation leads to insulin resistance. We previously reported that alterations in a monocyte cholesterol metabolism transcriptional network (CMTN) - suggestive of cellular cholesterol accumulation - were cross-sectionally associated with obesity and type 2 diabetes (T2D). Here, we sought to determine whether the CMTN alterations independently predict incident prediabetes/T2D risk, and correlate with cellular cholesterol accumulation.METHODSMonocyte mRNA expression of 11 CMTN genes was quantified among 934 Multi-Ethnic Study of Atherosclerosis (MESA) participants free of prediabetes/T2D; cellular cholesterol was measured in a subset of 24 monocyte samples.RESULTSDuring a median 6-year follow-up, lower expression of 3 highly correlated LXR target genes - ABCG1 and ABCA1 (cholesterol efflux) and MYLIP (cholesterol uptake suppression) - and not other CMTN genes, was significantly associated with higher risk of incident prediabetes/T2D. Lower expression of the LXR target genes correlated with higher cellular cholesterol levels (e.g., 47% of variance in cellular total cholesterol explained by ABCG1 expression). Further, adding the LXR target genes to overweight/obesity and other known predictors significantly improved prediction of incident prediabetes/T2D.CONCLUSIONThese data suggest that the aberrant LXR/ABCG1-ABCA1-MYLIP pathway (LAAMP) is a major T2D risk factor and support a potential role for aberrant LAAMP and cellular cholesterol accumulation in diabetogenesis.FUNDINGThe MESA Epigenomics and Transcriptomics Studies were funded by NIH grants 1R01HL101250, 1RF1AG054474, R01HL126477, R01DK101921, and R01HL135009. This work was supported by funding from NIDDK R01DK103531 and NHLBI R01HL119962.

A New Autosomal Myh11-CreERT2 Smooth Muscle Cell Lineage Tracing and Gene Knockout Mouse Model-Brief Report.

BACKGROUND: The Myh11 promoter is extensively used as a smooth muscle cell (SMC) Cre-driver and is regarded as the most restrictive and specific promoter available to study SMCs. Unfortunately, in the existing Myh11-CreERT2 mouse, the transgene was inserted on the Y chromosome precluding the study of female mice. Given the importance of including sex as a biological variable and that numerous SMC-based diseases have a sex-dependent bias, the field has been tremendously limited by the lack of a model to study both sexes. Here, we describe a new autosomal Myh11-CreERT2 mouse (referred to as Myh11-CreERT2-RAD), which allows for SMC-specific lineage tracing and gene knockout studies in vivo using both male and female mice. METHODS: A Myh11-CreERT2-RAD transgenic C57BL/6 mouse line was generated using bacterial artificial chromosome clone RP23-151J22 modified to contain a Cre-ERT2 after the Myh11 start codon. Myh11-CreERT2-RAD mice were crossed with 2 different fluorescent reporter mice and tested for SMC-specific labeling by flow cytometric and immunofluorescence analyses. RESULTS: Myh11-CreERT2-RAD transgene insertion was determined to be on mouse chromosome 2. Myh11-CreERT2-RAD fluorescent reporter mice showed Cre-dependent, tamoxifen-inducible labeling of SMCs equivalent to the widely used Myh11-CreERT2 mice. Labeling was equivalent in both male and female Cre+ mice and was limited to vascular and visceral SMCs and pericytes in various tissues as assessed by immunofluorescence. CONCLUSIONS: We generated and validated the function of an autosomal Myh11-CreERT2-RAD mouse that can be used to assess sex as a biological variable with respect to the normal and pathophysiological functions of SMCs.

Monocyte miRNAs Are Associated With Type 2 Diabetes.

miRNAs are small noncoding RNAs that may contribute to common diseases through epigenetic regulation of gene expression. Little is known regarding the role of miRNAs in type 2 diabetes (T2D). We performed miRNA sequencing and transcriptomic profiling of peripheral monocytes from the longitudinal Multi-Ethnic Study of Atherosclerosis (MESA) (N = 1,154). We examined associations between miRNAs and prevalent impaired fasting glucose and T2D and evaluated the T2D-associated miRNA effect on incident T2D. Of 774 detected miRNAs, 6 (miR-22-3p, miR-33a-5p, miR-181c-5p, miR-92b-3p, miR-222-3p, and miR-944) were associated with prevalent T2D. For five of the six miRNAs (all but miR-222-3p), our findings suggest a dose-response relationship with impaired fasting glucose and T2D. Two of the six miRNAs were associated with incident T2D (miR-92b-3p: hazard ratio [HR] 1.64, P = 1.30E-03; miR-222-3p: HR 1.97, P = 9.10E-03) in the highest versus lowest tertile of expression. Most of the T2D-associated miRNAs were also associated with HDL cholesterol concentrations. The genes targeted by these miRNAs belong to key nodes of a cholesterol metabolism transcriptomic network. Higher levels of miRNA expression expected to increase intracellular cholesterol accumulation in monocytes are linked to an increase in T2D risk.

Scavenger Receptor CD36 Directs Nonclassical Monocyte Patrolling Along the Endothelium During Early Atherogenesis.

OBJECTIVE: Nonclassical monocytes (NCM) function to maintain vascular homeostasis by crawling or patrolling along the vessel wall. This subset of monocytes responds to viruses, tumor cells, and other pathogens to aid in protection of the host. In this study, we wished to determine how early atherogenesis impacts NCM patrolling in the vasculature. APPROACH AND RESULTS: To study the role of NCM in early atherogenesis, we quantified the patrolling behaviors of NCM in ApoE-/- (apolipoprotein E) and C57BL/6J mice fed a Western diet. Using intravital imaging, we found that NCM from Western diet-fed mice display a 4-fold increase in patrolling activity within large peripheral blood vessels. Both human and mouse NCM preferentially engulfed OxLDL (oxidized low-density lipoprotein) in the vasculature, and we observed that OxLDL selectively induced NCM patrolling in vivo. Induction of patrolling during early atherogenesis required scavenger receptor CD36, as CD36-/- mice revealed a significant reduction in patrolling activity along the femoral vasculature. Mechanistically, we found that CD36-regulated patrolling was mediated by a SFK (src family kinase) through DAP12 (DNAX activating protein of 12KDa) adaptor protein. CONCLUSIONS: Our studies show a novel pathway for induction of NCM patrolling along the vascular wall during early atherogenesis. Mice fed a Western diet showed increased NCM patrolling activity with a concurrent increase in SFK phosphorylation. This patrolling activity was lost in the absence of either CD36 or DAP12. These data suggest that NCM function in an atheroprotective manner through sensing and responding to oxidized lipoprotein moieties via scavenger receptor engagement during early atherogenesis.

Human Blood Monocyte Subsets: A New Gating Strategy Defined Using Cell Surface Markers Identified by Mass Cytometry.

OBJECTIVE: Human monocyte subsets are defined as classical (CD14++CD16-), intermediate (CD14++CD16+), and nonclassical (CD14+CD16+). Alterations in monocyte subset frequencies are associated with clinical outcomes, including cardiovascular disease, in which circulating intermediate monocytes independently predict cardiovascular events. However, delineating mechanisms of monocyte function is hampered by inconsistent results among studies. APPROACH AND RESULTS: We use cytometry by time-of-flight mass cytometry to profile human monocytes using a panel of 36 cell surface markers. Using the dimensionality reduction approach visual interactive stochastic neighbor embedding (viSNE), we define monocytes by incorporating all cell surface markers simultaneously. Using viSNE, we find that although classical monocytes are defined with high purity using CD14 and CD16, intermediate and nonclassical monocytes defined using CD14 and CD16 alone are frequently contaminated, with average intermediate and nonclassical monocyte purity of ≈86.0% and 87.2%, respectively. To improve the monocyte purity, we devised a new gating scheme that takes advantage of the shared coexpression of cell surface markers on each subset. In addition to CD14 and CD16, CCR2, CD36, HLA-DR, and CD11c are the most informative markers that discriminate among the 3 monocyte populations. Using these additional markers as filters, our revised gating scheme increases the purity of both intermediate and nonclassical monocyte subsets to 98.8% and 99.1%, respectively. We demonstrate the use of this new gating scheme using conventional flow cytometry of peripheral blood mononuclear cells from subjects with cardiovascular disease. CONCLUSIONS: Using cytometry by time-of-flight mass cytometry, we have identified a small panel of surface markers that can significantly improve monocyte subset identification and purity in flow cytometry. Such a revised gating scheme will be useful for clinical studies of monocyte function in human cardiovascular disease.

The diverse functions of Dot1 and H3K79 methylation.

DOT1 (disruptor of telomeric silencing; also called Kmt4) was initially discovered in budding yeast in a genetic screen for genes whose deletion confers defects in telomeric silencing. Since the discovery ∼10 years ago that Dot1 and its mammalian homolog, DOT1L (DOT1-Like), possess histone methyltransferase activity toward histone H3 Lys 79, great progress has been made in characterizing their enzymatic activities and the role of Dot1/DOT1L-mediated H3K79 methylation in transcriptional regulation, cell cycle regulation, and the DNA damage response. In addition, gene disruption in mice has revealed that mouse DOT1L plays an essential role in embryonic development, hematopoiesis, cardiac function, and the development of leukemia. The involvement of DOT1L enzymatic activity in leukemogenesis driven by a subset of MLL (mixed-lineage leukemia) fusion proteins raises the possibility of targeting DOT1L for therapeutic intervention.

DOT1L, the H3K79 methyltransferase, is required for MLL-AF9-mediated leukemogenesis.

Chromosomal translocations of the mixed lineage leukemia (MLL) gene are a common cause of acute leukemias. The oncogenic function of MLL fusion proteins is, in part, mediated through aberrant activation of Hoxa genes and Meis1, among others. Here we demonstrate using a tamoxifen-inducible Cre-mediated loss of function mouse model that DOT1L, an H3K79 methyltransferase, is required for both initiation and maintenance of MLL-AF9-induced leukemogenesis in vitro and in vivo. Through gene expression and chromatin immunoprecipitation analysis we demonstrate that mistargeting of DOT1L, subsequent H3K79 methylation, and up-regulation of Hoxa and Meis1 genes underlie the molecular mechanism of how DOT1L contributes to MLL-AF9-mediated leukemogenesis. Our study not only provides the first in vivo evidence for the function of DOT1L in leukemia, but also reveals the molecular mechanism for DOT1L in MLL-AF9 mediated leukemia. Thus, DOT1L may serve as a potential therapeutic target for the treatment of leukemia caused by MLL translocations.

KDM2b/JHDM1b, an H3K36me2-specific demethylase, is required for initiation and maintenance of acute myeloid leukemia.

The histone H3 lysine 36 dimethyl-specific demethylase KDM2b/JHDM1b, which is highly expressed in various human leukemias, was previously found to be important in regulating cell proliferation and cellular senescence. However, its functions in leukemia development and maintenance are unclear. Here, we demonstrate that ectopic expression of Kdm2b/Jhdm1b is sufficient to transform hematopoietic progenitors. Conversely, depletion of Kdm2b/Jhdm1b in hematopoietic progenitors significantly impairs Hoxa9/Meis1-induced leukemic transformation. In leukemic stem cells, knockdown of Kdm2b/Jhdm1b impairs their self-renewing capability in vitro and in vivo. The functions of Kdm2b/Jhdm1b are mediated by its silencing of p15(Ink4b) expression through active demethylation of histone H3 lysine 36 dimethyl. Thus, our study suggests that Kdm2b/Jhdm1b functions as an oncogene and plays a critical role in leukemia development and maintenance.

Antibody-functionalized polydiacetylene coatings on nanoporous membranes for microorganism detection.

The preparation and characterization of coatings made from polydiacetylene colloids on nano- and microporous membranes and their potential for the detection of microorganisms are presented.