Effect of apoE enrichment on lipoprotein cholesterol profile.

<p>Fifty µg of apoE-free (E⁻) lipoproteins (<1.063) were incubated with 5 µg of apoE3 or apoE4 or an equivalent volume of vehicle at RT for 1 h. Cholesterol profiles in E^- lipoproteins (A), apoE3- (B), and apoE4-enriched (C) lipoproteins were analyzed with fast protein liquid chromatography.</p

The effect of ApoE on lipoprotein-induced Sp1 DNA-binding activity.

<p>Mouse macrophages were treated with 20 µg/ml of wild-type (E⁺/B), ApoE-free (EÎ/B) or ApoE3-enriched (E3/B) lipoproteins, or culture medium alone (control) for 4 hrs. The amount of Sp1 bound to the ABCA1 promoter region was determined by ChIP analysis, and expressed as the ratio of input controls. Values represent the mean ± SEM of 5 separate experiments. * <i>P</i><0.05 compared to control, ^†<i>P</i><0.05 compared to E⁺/B or E3/B lipoprotein treatment.</p

Effect of lipid-free apoE isoforms on phosphorylation of PKCζ and Sp1, and ABCA1 expression in human THP-1 macrophages.

<p>(A–D) Human macrophage THP-1 cells were treated with 3 µg/ml of lipid-free apoEs for as described under Materials and Methods and the protein levels of ABCA1, p-PKCζ and Sp1 determined by immunoblotting. *<i>P</i><0.05 or **<i>P</i><0.01 relative to controls; ^#<i>P</i><0.05 relative to apoE3.</p

Effect of lipoprotein-associated apoE isoforms on PI3K and PKCζ phosphorylation.

<p>RAW 264.7 macrophages were incubated at 37°C for 5 h with 20 µg/ml of apoE-free (E⁻) lipoprotein, 20 µg/ml of E⁻ lipoproteins containing 2 µg/ml of apoE3 (E3) or apoE4 (E4) or culture medium alone as a control (Ctrl). (A–C) Phosphorylated PI3K and PKCζ were determined by immunoblotting. Data represent the mean ± SEM from 3–4 separate experiments. *<i>P</i><0.05 or **<i>P</i><0.01 relative to controls; ^†<i>P</i><0.05 relative to E⁻; <b>^#</b><i>P</i><0.05 relative to apoE3.</p

The effect of mithramycin A on lipoprotein-induced ABCA1 expression and cholesterol efflux.

<p>For study ABCA1 expression, mouse macrophages were treated with 20 µg/ml of wild-type (E⁺/B), ApoE-free (EÎ/B) or ApoE3-enriched (E3/B) lipoproteins, or culture medium (control) in the presence or absence of 100 nM mithramycin A (mmA) for 4 hrs. <b>Panel A and B:</b> The level of ABCA1 protein was determined by western blot analysis and quantitated relative to β-actin. <b>Panel C:</b> The level of ABCA1 mRNA was determined by quantitative real-time RT-PCR and normalized to GAPDH mRNA. <b>Panel D:</b> For studying cholesterol efflux, mouse macrophages were incubated with [³H]-cholesterol in the presence or absence of 20 µg/ml of E⁺/B, E⁻/B or E3/B lipoproteins for 24 hrs. ApoAI-mediated cholesterol efflux was measured in the presence or absence of mmA. Values represent the mean ± SEM of five independent experiments. * <i>P</i><0.05 compared to control, ^†<i>P</i><0.05 compared to cells treated with E⁺/B lipoproteins and without mmA, and ^#<i>P</i><0.05 compared to cells treated with E⁻/B lipoproteins and without mmA.</p

Effect of lipid-free apoE and apoAI on cholesterol efflux.

<p>(A–B) RAW 264.7 macrophages were incubated at 37°C for 48 h with 1 µCi/ml of ³H-cholesterol and 20 µg/ml apoE-free (E⁻) lipoprotein, followed by incubation with the indicated amount of apoE3 (E3) or apoE4 (E4) for an additional 5 h. Thereafter, cells were incubated with 20 µg/ml of apoAI or base medium alone for 2 h. Cholesterol efflux was determined as described in the Materials and Methods. ApoAI mediated cholesterol efflux was calculated as the total efflux in the presence of apoAI minus the efflux to the base medium without apoAI. (C) The cells were loaded with ³H-cholesterol as abovementioned, and then incubated with 0.44 µM of lipid free apoA1 (12.35 µg/ml), apoE3 or apoE4 (15 µg/ml), or culture medium alone as a control (ctrl) for 6 h. Total cholesterol efflux was determined as described in the Materials and Methods. Data represent the mean ± SEM from 3–4 separate experiments. *<i>P</i><0.05 or **<i>P</i><0.01 relative to controls; <b>^#</b><i>P</i><0.05 relative to apoE4.</p

Effect of lipid-associated apoE isoforms on Sp1 expression and phosphorylation.

<p>(A) RAW 264.7 cell lysates were incubated for 5 h with or without λ-phosphatase and Sp1 levels determined by immunoblotting. The upper band (p-Sp1) was determined to be the phosphorylated form (p-Sp1) and the lower band (unp-Sp1), the unphosphorylated form. (B–E) RAW 264.7 macrophages were incubated at 37°C for 5 h with 20 µg/ml of apoE-free (E⁻) lipoprotein, 20 µg/ml of E⁻ lipoproteins containing 2 µg/ml of apoE3 (E3) or apoE4 (E4) or culture medium alone (Ctrl). The level of phosphorylated Sp1 (top band) was expressed relative to the total Sp1 protein level (the sum of the top and bottom bands relative to actin) or actin. Data represent the mean ± SEM from 3–4 separate experiments. *<i>P</i><0.05 or **<i>P</i><0.01 relative to controls; ^†<i>P</i><0.05 relative to E⁻; <b>^#</b><i>P</i><0.05 relative to apoE3.</p

Effect of lipid-free apoE isoforms on Sp1 expression and phosphorylation.

<p>(A–D) RAW 264.7 macrophages were incubated at 37°C for 5 h with 3 µg/ml of lipid-free apoE3 (E3) or apoE4 (E4) or culture medium alone as a control (Ctrl). Sp1 protein was determined by immunoblotting. The level of phosphorylated Sp1 (top band) was expressed relative to the total Sp1 protein level (the sum of the top and bottom bands relative to actin) or actin. Data represent the mean ± SEM from 3–4 separate experiments. *P<0.05 or **P<0.01 relative to controls; <b>^#</b>P<0.05 relative to apoE3.</p

The effect of ApoE on lipoprotein-induced Sp1 phosphorylation.

<p>Mouse macrophages were treated with 20 µg/ml of wild-type (E⁺/B), ApoE-free (EÎ/B) or ApoE3-enriched (E3/B) lipoproteins, or culture medium alone (control) for 4 hrs. The cell lysate were incubated with or without λ-phosphatase. Sp1 phosphorylation was detected by Sp1 immunoblot band shift induced by lipoprotein and λ-phosphatase treatments. The level of phosphorylated Sp1 was expressed as the percentage of the immunoreactive intensity of the top band versus the total (top plus bottom bands). Values represent the mean ± SEM of 3 separate experiments. * <i>P</i><0.05 compared to control, ^†<i>P</i><0.05 compared to E⁺ or E3 lipoprotein treatment.</p

DataSheet_1_Prediction model based on preoperative CT findings for carotid artery invasion in patients with head and neck masses.pdf

ObjectivesTo investigate the performance of a model in predicting carotid artery (CA) invasion in patients with head and neck masses using computed tomography (CT).MethodsThis retrospective study included patients with head and neck masses who underwent CT and surgery between January 2013 and July 2021. Patient characteristics and ten CT features were assessed by two radiologists. The patients were randomly allocated to a training cohort (n=106) and a validation cohort (n=109). Independent risk factors for CA invasion were assessed by univariate and multivariate logistic regression analyses. The predictive model was established as a nomogram using the training cohort. In addition, the calibration, discrimination, reclassification, and clinical application of the model were assessed in the validation cohort.ResultsA total of 215 patients were evaluated, including 54 patients with CA invasion. Vascular wall deformation (odds ratio [OR], 7.17; p=0.02) and the extent of encasement to the CA (OR, 1.02; pConclusionA predictive model for carotid artery invasion can be defined based on features that come from patient characteristics and CT data to help in improve surgical planning and invasion evaluation.</p