Potential Biomarker Peptides Associated with Acute Alcohol-Induced Reduction of Blood Pressure

<div><p>The purpose of this study was to explore the peptides that are related to acute reduction of blood pressure after alcohol drinking. Venous blood was collected from male healthy volunteers before and after drinking white wine (3 ml/kg weight) containing 13% of ethanol. Peptidome analysis for serum samples was performed using a new target plate, BLOTCHIP^®. Alcohol caused significant decreases in systolic and diastolic blood pressure levels at 45 min. The peptidome analysis showed that the levels of three peptides of <i>m/z</i> 1467, 2380 and 2662 changed significantly after drinking. The <i>m/z</i> 1467 and 2662 peptides were identified to be fragments of fibrinogen alpha chain, and the <i>m/z</i> 2380 peptide was identified to be a fragment of complement C4. The intensities of the <i>m/z</i> 2380 and <i>m/z</i> 1467 peptides before drinking were associated with % decreases in systolic and diastolic blood pressure levels at 45 min after drinking compared with the levels before drinking, while there were no significant correlations between the intensity of the <i>m/z</i> 2662 peptide and % decreases in systolic and diastolic blood pressure levels after drinking. The <i>m/z</i> 1467 and 2380 peptides are suggested to be markers for acute reduction of blood pressure after drinking alcohol.</p></div

Differential profiling of serum sampled from subjects before and after drinking.

<p>Each integrated spectrum normalized with ClinPro Tools version 2.2 is the average of 10 samples (total of 44 integrations) from subjects.</p

Scatter plots of the relationships between ranks of serum <i>m/z</i> 2380 peptide intensity before drinking and % changes in systolic (A) and diastolic (B) blood pressure at 45 min after drinking.

<p>Spearman’s rank correlation coefficients (<i>r</i>) are given in the figures.</p

MS/MS structural analysis of <i>m/z</i> 1467 (A), 2380 (B), 2662 (C) peptides in the sample after reversed-phase high-pressure liquid chromatography separation.

<p>MS/MS structural analysis of <i>m/z</i> 1467 (A), 2380 (B), 2662 (C) peptides in the sample after reversed-phase high-pressure liquid chromatography separation.</p

Changes in systolic (A) and diastolic (B) blood pressure levels after drinking alcohol.

<p>Blood pressure was measured just before dinking and at 45 min and 2–3 hr after drinking. Means ± standard errors of blood pressure levels are shown. Asterisks denote significant differences from the levels before drinking (**, <i>p</i> < 0.01).</p

Additional file 1 of NG2-positive pericytes regulate homeostatic maintenance of slow-type skeletal muscle with rapid myonuclear turnover

Additional file 1: Fig. S1. Localization of NG2+ cells in adult skeletal muscle tissues. Circulating vessels in NG2-DsRed mice were visualized by intravenous injection of FITC-conjugated lectin. The lower limb skeletal muscles (gastrocnemius and soleus) were fixed and transparentized with RapiClear reagent. Microvessels, lectin-labeled endothelium tubes (lectin; green), and NG2+ cells (DsRed; red) within transparent muscles were visualized in a 3D view using confocal fluorescent microscopy. The nuclei were counterstained with DAPI. Scale bar = 50 µm. Fig. S2. NG2+ cell lineage tracing within the skeletal muscle. The tdTomato expression driven by the universal Rosa26 promoter was specifically induced in NG2+ cells using NG2-CreERT/Rosa-tdTomato mice. After five days of constitutive treatment with Tam, NG2+ cells were expressed. B. On day 1 of the observation period, tdTomato+ cells were observed only at perivascular sites, such as PCs. On day 21, tdTomato-expressing myofibers were observed in most muscle tissues. The ratio of tdTomato+ myofibers to total myofibers varied by muscle site, i.e., over 80% of tdTomato+ myofibers in the soleus and diaphragm and 20–30% in the gastrocnemius and rectus abdominal muscles. Scale bar = 200 µm. Fig. S3. Schematic diagram for the in vitro muscular differentiation assay. Myofibers were isolated from the soleus of NG2-CreERT/Rosa-tdTomato mice by using a collagenase-containing medium. Isolated myofibers were incubated in DMEM-containing 10% FBS and Tam (2 µM) for three days to label NG2+ PCs. The medium was then changed to a differentiation medium containing 2% horse serum. After six days of induction, the myogenesis of NG2+ PCs was observed. Fig. S4. In vitro myogenic potency of NG2+ PCs from soleus muscles. A. Myofibers isolated from the soleus of NG2-CreERT/Rosa-tdTomato mice, which were incubated in DMEM-containing hydroxy tamoxifen (Tam) for three days to label NG2+ PCs. After six days of differentiation induction, myogenesis was determined by immunostaining with myosin heavy chain (MyHC) and myosin heavy chain (MYH) isoform 2 and 7. Isolated soleus myofibers were used for control for immunostaining. Scale bars = 100 µm. Fig. S5. Tam treatment induces deletion of NG2+ cells. A. In vitro effects of 4-hydroxytamoxifen (4-HT) on NG2+ cells isolated from subcutaneous adipose tissues of NG2-CreERT/Rosa26-DTA mice. Cells at confluent were incubated in medium containing hydroxy-Tam for 5 days, and the number of cells were counted. B. Gene expression of NG2 in NG2+ cells with Tam was esteemed by qPCR. Fig. S6. Microarray enrichment analysis in response to PC deletion. After induction of NG2+ PC deletion for one month, microarray analysis of the soleus of PC-deletion and control mice was performed. The top 20 upregulated and downregulated pathway-related gene sets are listed