PLoS ONE 11(8): e0160530. doi:10.1371/journal.pone.0160530

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

 

Authors: Yoshiro Saito and Noriko Noguchi 

Department of Medical Life Systems, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Kyoto 610–0321, Japan

 

Address for correspondence:

Yoshiro Saito, PhD

Department of Medical Life Systems, Faculty of Life and Medical Sciences, Doshisha University, 1-3 Miyakodani, Tatara, Kyotanabe, Kyoto 610–0394, Japan

Tel: +81-774-65-6258. E-mail: ysaito@mail.doshisha.ac.jp

 

Abstract

Backgrounds: 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.  To clarify the role of serum oxidation products in vessel cell proliferation, the major vessel cell proliferator in oxidized human serum was investigated.

Methods and Results: 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.  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.

Conclusions: Oxidized lipoproteins containing lipid oxidation products function as a major vessel cell proliferator in oxidized human serum.

PMID: 27483438

 

Supplementary

The relationship between oxidative stress biomarkers and their biological action

The oxidation of biological molecules by free radicals yields a variety of oxidation products [1].  Oxidized products are measured as biomarkers of oxidative stress to assess the oxidative injury in the pathologic processes of free radical-related diseases [2].  However, the relationship between oxidative stress biomarkers and their biological action has not been well investigated.  The proliferation of vascular cells is related to the onset as well as the progress of several diseases such as atherosclerosis and cancer.  To clarify the role of serum oxidation products in vessel cell proliferation, the major vessel cell proliferator in oxidized human serum was investigated.

 

Effects of oxidized human serum components on the viability of vessel cells

To examine the effects of oxidized products in serum, a water-soluble radical initiator was added to human serum, and the serum component was oxidized [3].  The increase in cholesteryl ester hydroperoxide (CE-OOH), a major lipid peroxidation product in serum, and protein oxidation products (protein carbonyl) was confirmed [4].  Oxidized human serum was applied to gel chromatography, and then each fraction was added to the culture media of vessel cells such as endothelial cells (ECs) and smooth muscle cells (SMCs). After 48 h, cell viability was determined.  No statistically significant change was observed in the component of control human serum (Fig 1A).  In contrast, a significant increase in human aortic endothelial cells (HAEC) viability was observed in the case of oxidized human serum, in which two peaks are observed: one is a macromolecule of more than 200 kDa at fraction number 12 (Fr. 12) and the other is of low molecular weight at Fr. 36 (Fig 1B).  In the other types of vessel cells such as human aortic smooth muscle cells (AoSMCs) and human umbilical vein endothelial cells (HUVECs), a statistically significant increase was observed in macromolecule peaks at Fr. 12 (Fig 1C).  A significant increase in vessel cell viability with a single peak at around 200 kDa was observed in gel chromatography with a larger pore size (Fig 1E).  These results suggest that the macromolecule proliferator for vessel cells is generated in oxidized human serum.

 

Distribution of oxidized products in gel chromatography

The distribution of oxidized products, such as CE-OOH and protein carbonyls, in gel chromatography of human oxidized serum was analyzed. The peak of CE-OOH was consistent with that of the macromolecular proliferator for vessel cells in oxidized human serum (Fig 2A and 2B).  CE-OOH mainly exists in lipoproteins. Thus, these results suggest that oxidized lipoproteins containing CE-OOH act as the macromolecular proliferator for vessel cells in oxidized human serum.

 

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

To investigate the involvement of oxidized lipoproteins, lipoprotein-deficient serum (LPDS) was prepared and oxidized by peroxyl radical initiator.  It was found that the significant increase in cell viability of ECs and SMCs disappeared in a high molecular weight fraction of oxidized LPDS (Fig 3). In contrast, HAEC viability increased at Fr. 12 when cultured with oxidized human serum components (Fig 3 inset). Collectively, these results suggest that the activity of the macromolecular proliferator for vessel cells in oxidized human serum is derived from oxidized lipoproteins.

 

Oxidized lipoprotein as a major vessel cell proliferator in oxidized human serum

In the present study using oxidized human serum, we could identify oxidized lipoproteins, containing CE-OOH, as a major proliferator of vascular cells (Fig. 4). This result clearly suggests the relationship between oxidative stress and the proliferation of vessel cells, which are related to the progression of arteriosclerosis and cancer, and suggests the significance of oxidized lipoproteins and lipids in the evaluation of the pathogenesis of atherosclerosis and cancer.

 

References

[1] Niki E, Yoshida Y, Saito Y, Noguchi N. Lipid peroxidation: mechanisms, inhibition, and biological effects. Biochem Biophys Res Commun. 2005;338(1):668–76. pmid:16126168

[2] Saito Y, Shichiri M, Hamajima T, Ishida N, Mita Y, Nakao S, et al. Enhancement of lipid peroxidation and its amelioration by vitamin E in a subject with mutations in the SBP2 gene. J Lipid Res. 2015;56(11):2172–82. doi: 10.1194/jlr.M059105. pmid:26411970

[3] Yoshida Y, Itoh N, Saito Y, Hayakawa M, Niki E. Application of water-soluble radical initiator, 2,2′-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride, to a study of oxidative stress. Free Rad Res. 2004;38(4):375–84.

[4] Saito Y, Noguchi N. 7-Hydroxycholestrol as a possible biomarker of cellular lipid peroxidation: difference between cellular and plasma lipid peroxidation. Biochem Biophys Res Commun. 2014;446(3):741–4. doi: 10.1016/j.bbrc.2013.12.083. pmid:24380859

 

 

Figure legend

 

Fig 1. Effects of oxidized human serum components on vessel cell viability.

A-D. Control (A) and oxidized human serum (B, C) were fractionated by Sephacryl S-300 gel chromatography, and protein contents were determined by absorbance at 280 nm.  HAEC (A, B), AoSMC (C), and HUVEC (D) were treated with each fraction (10%) for 48h, and the viability was measured.  The maclomolecule proliferator for vessel cells is indicated by black arrow. D. 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. * P < 0.01, compared with vehicle control.

 

 

 

Fig 2. Distribution of oxidized products in Sephacryl S-300 gel chromatography.

A. 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. 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.

 

 

Fig 3. Effects of oxidized lipoprotein deficient serum on the viability of vessel cells.

A and 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 (A) and AoSMC (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. The maclomolecule proliferator for vessel cells at Fr.12 is indicated by black arrow. * P < 0.01, compared with vehicle control.

 

 

 

Fig 4. Oxidized lipoprotein as a major vessel cell proliferator in oxidized human serum.

This study could identify oxidized lipoproteins as a major proliferator of vascular cells in human serum treated with radical initiator, and indicates the significance for the evaluation of oxidized lipoproteins and lipids in atherosclerosis and cancer patients to assume disorder of vessel cells.

 

 

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