Oxidized Lipoprotein as a Major Vessel Cell Proliferator in Oxidized Human Serum

<div><p>Oxidative stress is correlated with the incidence of several diseases such as atherosclerosis and cancer, and oxidized biomolecules have been determined as biomarkers of oxidative stress; however, the detailed molecular relationship between generated oxidation products and the promotion of diseases has not been fully elucidated. In the present study, to clarify the role of serum oxidation products in vessel cell proliferation, which is related to the incidence of atherosclerosis and cancer, the major vessel cell proliferator in oxidized human serum was investigated. Oxidized human serum was prepared by free radical exposure, separated using gel chromatography, and then each fraction was added to several kinds of vessel cells including endothelial cells and smooth muscle cells. It was found that a high molecular weight fraction in oxidized human serum specifically induced vessel cell proliferation. Oxidized lipids were contained in this high molecular weight fraction, while cell proliferation activity was not observed in oxidized lipoprotein-deficient serum. Oxidized low-density lipoproteins induced vessel cell proliferation in a concentration-dependent manner. Taken together, these results indicate that oxidized lipoproteins containing lipid oxidation products function as a major vessel cell proliferator in oxidized human serum. These findings strongly indicate the relevance of determination of oxidized lipoproteins and lipid oxidation products in the diagnosis of vessel cell proliferation-related diseases such as atherosclerosis and cancer.</p></div

Effects of oxidized low density lipoprotein on the viability of vessel cells.

<p>HUVEC was treated with variable concentrations of oxidized LDL for 48h, and the viability was measured by WST assay. * <i>P</i> < 0.01, compared with vehicle control.</p

Effects of oxidized human serum components on vessel cell viability.

<p><b>A-D.</b> Control (<b>A</b>) and oxidized human serum (<b>B-D</b>) were fractionated by Sephacryl S-300 gel chromatography, and protein contents were determined by absorbance at 280 nm. HAEC (<b>A, B</b>), AoSMC (<b>C</b>), and HUVEC (<b>D</b>) were treated with each fraction (10%) for 48h, and the viability was measured by WST assay, as described under “Materials and methods”. The maclomolecule proliferator for vessel cells at Fr.12 is indicated by black arrow. <b>E.</b> Oxidized human serum were fractionated by Sephacryl S-500 gel chromatography, and HUVEC was treated each fraction for 48h. The maclomolecule proliferator for vessel cells at Fr.31 is indicated by black arrow. * <i>P</i> < 0.01, compared with vehicle control.</p

Contents of lipid peroxidation products, protein carbonyl, and lipids in human serum samples used in this study^a.

<p>Contents of lipid peroxidation products, protein carbonyl, and lipids in human serum samples used in this study<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160530#t001fn002" target="_blank">^a</a>.</p

Distribution of oxidized products in Sephacryl S-300 gel chromatography.

<p><b>A.</b> Oxidized human serum were fractionated by Sephacryl S-300 gel chromatography, and contents of protein (A280), cholesterol ester hydroperoxide (CE-OOH), and protein carbonyl were determined, respectively. <b>B.</b> Both AoSMC viability and CE-OOH contents are plotted. The peak of maclomolecule proliferator for vessel cells and CE-OOH at Fr.12 is indicated by black arrow.</p

Effects of oxidized lipoprotein deficient serum on the viability of vessel cells.

<p><b>A-C.</b> Oxidized lipoprotein deficient serum (LPDS) were fractionated by Sephacryl S-300 gel chromatography, and protein contents were determined by absorbance at 280 nm. HAEC (<b>A</b>), AoSMC (<b>B</b>), and HUVEC (<b>C</b>) were treated with each fraction (10%) for 48h, and the viability was measured by WST assay. The cell viability in the case of oxidized human serum fractions are shown in inner figure. <b>D.</b> LPDS and extracted lipid fraction (L) were mixed, oxidized, and then fractionated by Sephacryl S-300 gel chromatography. HAEC was treated with each fraction (10%) for 48h, and the viability was measured. The maclomolecule proliferator for vessel cells at Fr.12 is indicated by black arrow. * <i>P</i> < 0.01, compared with vehicle control.</p

Plasma Lipid Profiling of Patients with Chronic Ocular Complications Caused by Stevens-Johnson Syndrome/Toxic Epidermal Necrolysis

<div><p>Stevens-Johnson syndrome (SJS) and its severe variant, toxic epidermal necrolysis (TEN), are drug-induced acute inflammatory vesiculobullous reactions of the skin and mucous membranes, including the ocular surface. Even after recovery from skin symptoms, some SJS/TEN patients continue to suffer with severe ocular complications (SOCs). Therefore, this study aims to understand the pathophysiology of chronic SOCs. Because plasma lipid profiling has emerged as a useful tool to understand pathophysiological alterations in the body, we performed plasma lipid profiling of 17 patients who suffered from SJS/TEN-associated chronic SOCs. A lipidomics approach yielded 386 lipid molecules and demonstrated that plasma levels of inflammatory oxylipins increased in patients with SJS/TEN-associated chronic SOCs. In addition, oxidized phosphatidylcholines and ether-type diacylglycerols increased in the patients with chronic SOCs, while phosphoglycerolipids decreased. When we compared these lipidomic profiles with those of patients with atopic dermatitis, we found that patients with chronic SOCs, specifically, had decreased levels of ether-type phosphatidylcholines (ePCs) containing arachidonic acid (AA), such as PC(18:0e/20:4) and PC(20:0e/20:4). To confirm our finding, we recruited additional patients, who suffered from SOC associated with SJS/TEN (up to 51 patients), and validated the decreased plasma levels of AA-containing ePCs. Our study provides insight into the alterations of plasma lipidomic profiles in chronic SOCs and into the pathophysiology of SJS/TEN-associated chronic SOCs.</p></div

Cross-Classification of Human Urinary Lipidome by Sex, Age, and Body Mass Index

<div><p>Technological advancements in past decades have led to the development of integrative analytical approaches to lipidomics, such as liquid chromatography-mass spectrometry (LC/MS), and information about biogenic lipids is rapidly accumulating. Although several cohort-based studies have been conducted on the composition of urinary lipidome, the data on urinary lipids cross-classified by sex, age, and body mass index (BMI) are insufficient to screen for various abnormalities. To promote the development of urinary lipid metabolome-based diagnostic assay, we analyzed 60 urine samples from healthy white adults (young (c.a., 30 years) and old (c.a., 60 years) men/women) using LC/MS. Women had a higher urinary concentration of omega-3 12-lipoxygenase (LOX)-generated oxylipins with anti-inflammatory activity compared to men. In addition, young women showed increased abundance of poly-unsaturated fatty acids (PUFAs) and cytochrome P450 (P450)-produced oxylipins with anti-hypertensive activity compared with young men, whereas elderly women exhibited higher concentration of 5-LOX-generated anti-inflammatory oxylipins than elderly men. There were no significant differences in urinary oxylipin levels between young and old subjects or between subjects with low and high BMI. Our findings suggest that sex, but neither ages nor BMI could be a confounding factor for measuring the composition of urinary lipid metabolites in the healthy population. The information showed contribute to the development of reliable biomarker findings from urine.</p></div

Additional file 1: Table S1. of Comparison of circulating lipid profiles between fasting humans and three animal species used in preclinical studies: mice, rats and rabbits

Normalized peak height of phosphoglycerolipids, sphingolipids, and neutral lipids in the plasma of all the mammals. (XLSX 112 kb

Fatty acid side chains, calculated effect size, and statistically required sample size of specific polyunsaturated phosphoglycerolipids altered in SJS/TEN-associated chronic SOCs.

<p>Fatty acid side chains, calculated effect size, and statistically required sample size of specific polyunsaturated phosphoglycerolipids altered in SJS/TEN-associated chronic SOCs.</p