Mouse methylation profiles for leukocyte cell types, and estimation of leukocyte fractions in inflamed gastrointestinal DNA samples.

Precise analysis of tissue DNA and RNA samples is often hampered by contaminating non-target cells whose amounts are highly variable. DNA methylation profiles are specific to cell types, and can be utilized for assessment of the fraction of such contaminating non-target cells. Here, we aimed 1) to identify methylation profiles specific to multiple types of mouse leukocytes, and 2) to estimate the fraction of leukocytes infiltrating inflamed tissues using DNA samples. First, genome-wide DNA methylation analysis was conducted for three myeloid-lineage cells and four lymphoid-lineage cells isolated by fluorescence-activated cell sorting after magnetic-activated cell sorting from leukocytes in the spleen. Clustering analysis using CpG sites within enhancers separated the three myeloid-lineage cells and four lymphoid-lineage cells while that using promoter CpG islands (TSS200CGIs) did not. Among the 266,108 CpG sites analyzed, one CpG site was specifically hypermethylated (ß value ≥ 0.7) in B cells, and four, seven, 183, and 34 CpG sites were specifically hypomethylated (ß value < 0.2) in CD4+ T cells, CD8+ T cells, B cells, and NK cells, respectively. Importantly, cell type-specific hypomethylated CpG sites were located at genes involved in cell type-specific biological functions. Then, marker CpG sites to estimate the leukocyte fraction in a tissue with leukocyte infiltration were selected, and an estimation algorithm was established. The fractions of infiltrating leukocytes were estimated to be 1.6-12.4% in the stomach (n = 10) with Helicobacter pylori-induced inflammation and 1.5-4.3% in the colon with dextran sulfate sodium-induced colitis (n = 4), and the fractions were highly correlated with those estimated histologically using Cd45-stained tissue sections [R = 0.811 (p = 0.004)]. These results showed that mouse methylation profiles at CpG sites within enhancers reflected leukocyte cell lineages, and the use of marker CpG sites successfully estimated the leukocyte fraction in inflamed gastric and colon tissues.

Sleeping Beauty transposon mutagenesis identified genes and pathways involved in inflammation-associated colon tumor development.

Chronic inflammation promotes development and progression of colorectal cancer (CRC). To comprehensively understand the molecular mechanisms underlying the development and progression of inflamed CRC, we perform in vivo screening and identify 142 genes that are frequently mutated in inflammation-associated colon tumors. These genes include senescence and TGFß-activin signaling genes. We find that TNFα can induce stemness and activate senescence signaling by enhancing cell plasticity in colonic epithelial cells, which could act as a selective pressure to mutate senescence-related genes in inflammation-associated colonic tumors. Furthermore, we show the efficacy of the Cdk4/6 inhibitor in vivo for inflammation-associated colonic tumors. Finally, we functionally validate that Arhgap5 and Mecom are tumor suppressor genes, providing possible therapeutic targets for CRC. Thus, we demonstrate the importance of the inactivation of senescence pathways in CRC development and progression in an inflammatory microenvironment, which can help progress toward precision medicine.

The Utility of Intraventricular Pressure Gradient for Early Detection of Chemotherapy-Induced Subclinical Cardiac Dysfunction in Dogs.

Early detection of doxorubicin (DXR)-induced cardiomyopathy (DXR-ICM) is crucial to improve cancer patient outcomes and survival. In recent years, the intraventricular pressure gradient (IVPG) has been a breakthrough as a sensitive index to assess cardiac function. This study aimed to evaluate the usefulness of IVPG for the early detection of chemotherapy-related cardiac dysfunction. For this purpose, six dogs underwent conventional, speckle tracking, and color M-mode echocardiography concomitantly with pressure-and-volume analysis by conductance catheter. The cardiac function measurements were assessed before DXR administration (baseline, Pre), at the end of treatment protocol (Post), and at 1.5 years follow-up (Post2). The result showed a significant reduction in the left ventricular end-systolic pressure-volume (Emax: 4.4 ± 0.7, 6.1 ± 1.6 vs. 8.4 ± 0.8 mmHg/mL), total-IVPG (0.59 ± 0.12, 0.62 ± 0.15 vs. 0.86 ± 0.12 mmHg), and mid-IVPG (0.28 ± 0.12, 0.31 ± 0.11 vs. 0.48 ± 0.08 mmHg), respectively in Post2 and Post compared with the baseline (p < 0.05). Mid-to-apical IVPG was also reduced in Post2 compared with the baseline (0.29 ± 0.13 vs. 0.51 ± 0.11). Meanwhile, the fraction shortening, ejection fraction, and longitudinal strain revealed no change between groups. Total and mid-IVPG were significantly correlated with Emax (R = 0.49; p < 0.05, both) but only mid-IVPG was a predictor for Emax (R2 = 0.238, p = 0.040). In conclusion, this study revealed that impairment of contractility was the initial changes observed with DXR-ICM in dogs and only IVPG could noninvasively detect subclinical alterations in cardiac function. Color M-mode echocardiography-derived IVPG could be a potential marker for the early detection of doxorubicin cardiomyopathy.

Changes in left ventricular blood flow during diastole due to differences in chamber size in healthy dogs.

Vorticity is a novel index that reflects diastolic function of left ventricle. The size of the ventricle can influence the ventricular diastolic blood flow. We evaluated effect of ventricular size on diastolic function and diastolic intracardiac blood flow using a particular species of dogs, which has a wide range of body size. Vector flow mapping was used for evaluation of intracardiac blood flow, and intraventricular pressure gradient (IVPG) was used for evaluation of diastolic function. 58 dogs weighing 1.3-42.3 kg were included in this study. Vorticity was found to be inversely proportional to the length of the ventricular chamber. Intraventricular pressure difference was positively correlated with the length of the left ventricle, whereas IVPG was not. This study showed that the vorticity is influenced by the size of the left ventricle independently of other factors. To evaluate the hemodynamic state of each individual appropriately by using vorticity and IVPD, ventricular size should be taken into account especially in the field of veterinary medicine and human pediatric and adolescent cardiology.

Left ventricular vortex and intraventricular pressure difference in dogs under various loading conditions.

Restrictions on the conventional evaluation of diastolic function have been recognized, especially under various loading conditions. Recently, new noninvasive ventricular vortex indexes have been introduced and are expected to reflect the cardiac function. Physiologically, there is a hypothesis that the intraventricular pressure difference (IVPD) is related to the formation of vortexes. IVPD and vortex indexes were simultaneously measured, and the relationship between the two was investigated. To verify the possibility of diastolic vorticity as an index of diastolic relaxation, a correlation between diastolic vorticity and the load dependency of vorticity [time constant (τ)] was examined. Six healthy dogs were studied using transthoracic echocardiography, pressure, and a conductance catheter. Vorticity was analyzed using vector flow mapping (VFM). IVPD was determined using Euler's equation with color M-mode Doppler images. Data were obtained at baseline, at balloon dilatation in the thoracic aorta to alter afterload, at hydroxyethyl starch infusion to alter preload, and at milrinone administration to alter ventricular relaxation. Peak vorticity at early diastole (E-Vor) and IVPD of the midventricle (MIVPD) decreased under pressure loading, were unchanged under volume loading, and increased during milrinone administration. In multivariate analysis, the independent predictors of τ were global longitudinal strain, strain rate at early diastole, and E-Vor. MIVPD was strongly correlated with E-Vor ( r = 0.84). VFM-derived peak E vorticity was strongly related to IVPD, especially MIVPD, under various loading conditions. Both of these novel indexes are promising as reliable indexes of ventricular relaxation, independent from preload. NEW & NOTEWORTHY We showed the close relationship of vortex and intraventricular pressure difference and showed that both of them can become new markers of the left ventricular relaxation property. Our present study creates a paradigm for future studies in the field of intraventircular flow physiology and clinical diastology.