Alteration of Fatty Acid Composition of Phospholipids in EA.hy926 Endothelial Cells Is Reflected in Microvesicles during TNF-α Stimulation

Abstract

The fatty acid composition of phospholipids in the plasma membrane affects cell function; however, these findings have primarily been observed in vitro because there are no suitable tools for monitoring phospholipid fatty acid composition of the plasma membrane in vivo. In this study, we used elaidate as a trigger for altering fatty acid composition in phospholipids and determined whether such changes are reflected in microvesicles (MVs) released from the plasma membrane. Persistent exposure of the human endothelial cell line EA.hy926 to elaidate altered the intracellular fatty acid composition of phospholipids; however, the changes occurring in whole cells were not reflected in the MV fraction during the basal state. Following stimulation with tumor necrosis factor-α, elaidate-mediated alterations in fatty acid composition of the intracellular phospholipids were observed in the MV fraction. Because the alteration of fatty acid composition of intracellular phospholipids is reflected in the plasma membrane, MVs may serve as a useful tool for monitoring the phospholipid fatty acid composition of the plasma membrane under limited conditions.

INTRODUCTION

Fatty acid is used not only as energy but also as cellular components. There are many types of fatty acids, and those balanced in phospholipids are involved in properties of the plasma membrane and various cell functions.¹⁾ However, these findings have been primarily observed in vitro,^2–4) as there are no less-invasive tools for monitoring the fatty acid composition of phospholipids in the plasma membrane in vivo.

Small vesicles released from cells into the blood may be used to monitor intracellular conditions. Although exosomes are used for monitoring intracellular proteins, lipids, and RNA, exosomes are released from exocytosis and do not contain plasma membrane phospholipids. Conversely, microvesicles (MVs) released from the plasma membrane contain proteins and phospholipids from the cell membrane.^5,6) Therefore, we hypothesized that MVs are a useful tool for monitoring the fatty acid composition of plasma membrane phospholipids. To monitor the effects of long-term alteration of blood fatty acid balance due to the daily diet on fatty acid composition of plasma membrane phospholipids in vivo, we examined the hypothesis using endothelial cells, which have a low turnover rate⁷⁾ and are directly exposed to blood. Moreover, although most blood MVs are derived from blood cells, endothelial-derived MVs account for several percent of the total blood MVs.⁸⁾

In this study, we used elaidate as a trigger for altering fatty acid composition in phospholipids of the plasma membrane⁹⁾ and determined whether the elaidate-induced changes in fatty acid composition are reflected in MVs.

MATERIALS AND METHODS

Cell Culture and Exposure to Elaidate

EA.hy926 endothelial cells were seeded at a density of 1.5 × 10⁵ cells per 60-mm dish and cultured for 4 d in Dulbecco’s modified Eagle’s medium containing 4.5 g/L d-glucose (Life Technologies, Carlsbad, CA, U.S.A.), 10% fetal bovine serum (FBS) (FB-1380; Biosera, Nuaille, France), 50 units/mL penicillin, and 50 µg/mL streptomycin (P7081; Sigma-Aldrich/Merck KGaA, Darmstadt, Germany).¹⁰⁾ The cells were reseeded at a density of 5 × 10⁵ cell per 100-mm dish and cultured for an additional 4 d. The cells were exposed to bovine serum albumin-conjugated elaidate¹¹⁾ continuously for 8 d. Control cells and the elaidate-exposed cells were stimulated with 10 ng/mL human recombinant tumor necrosis factor-α (TNF-α) (210-TA-005; R&D Systems, Minneapolis, MN, U.S.A.) for 24 h in the culture medium, which was centrifuged at 20000 × g for 30 min before use to remove any MVs in the FBS.

Preparation of MV Fraction

MVs were prepared from the culture supernatant by centrifugation as described previously.⁵⁾ Briefly, the culture supernatant was centrifuged at 2000 × g for 15 min at 4°C. After centrifuging the supernatant at 20000 × g for 15 min at 4°C, the pellet was resuspended in 20 mM N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid buffer (pH 7.4) containing 150 mM NaCl, and the suspension was further centrifuged at 20000 × g for 15 min at 4°C. The resulting pellet was collected as the MV fraction.

Analysis of Fatty Acid Composition of Phospholipids

The plasma membrane fraction was collected by centrifugation, followed by extraction of total lipids and isolation of phospholipids as previously described.^9,11) Isolated phospholipids were hydrolyzed and methylated, and the amount of fatty acid in each lipid was quantitated by GC-MS-QP2010 Ultra (SHIMADZU, Kyoto, Japan) using a Select FAME capillary column (200 m × 0.25 mm × 0.25 µm) (CP7421; Agilent Technologies, Santa Clara, CA, U.S.A.) in ion monitoring mode. The results were normalized to the amount of 1,2-diheptadecanoyl-sn-glycero-3-phosphorylcholine (No. 1400; Matreya LLC, Inc., Pleasant Gap, PA, U.S.A.) as an internal standard.

Measuring the Number of MVs by Flow Cytometry

The culture medium was centrifuged at 2000 × g for 15 min at 4°C. The resulting supernatant was diluted threefold with 140 mM NaCl and 2.5 mM CaC1₂ (Annexin V-binding buffer). Then, 3 µL of Annexin V-fluorescein isothiocyanate (4700-100; MBL, Nagoya, Japan) was added to 200 µL of the mixture and incubated for 15 min on ice. Annexin V-positive MVs were detected and counted using a BD FACS Aria III instrument (BD Biosciences, San Jose, CA, U.S.A.).

Statistical Analysis

Significant differences were calculated by one-way ANOVA followed by the Tukey–Kramer test using IBM SPSS Statistics (Version 28; IBM Japan, Tokyo, Japan), or tested for no correlation using Microsoft Excel 365. A p-value <0.05 was considered statistically significant.

RESULTS

Analysis of the Fatty Acid Composition of Phospholipids in Whole Cells and MV Fraction during the Basal State

The human endothelial cell line EA.hy926 was persistently exposed to a physiological concentration of elaidate for 8 d. The cells were further cultured with fresh medium for 24 h (Fig. 1A). Elaidate-exposed cells were collected, and the MV fraction was prepared from the culture supernatant. The fatty acid composition of phospholipids in whole cells was altered in a concentration-dependent manner by elaidate exposure (Fig. 1B); however, the fatty acid composition was not altered in the MV fraction by elaidate treatment (Fig. 1C). The relationship between the fatty acid composition of phospholipids in whole cells and the MV fraction was analyzed. We found that the ratio of most fatty acids, except for linoleate, was not correlated (Fig. 1D). Therefore, these results suggest that changes in the fatty acid composition of phospholipids in whole cells are not reflected in MVs released during the basal state.

Fig. 1. Analysis of the Fatty Acid Composition of Phospholipids in Whole Cells and the MV Fraction during the Basal State

(A) Schematic representation of the persistent exposure of EA.hy926 endothelial cells to elaidate. Fatty acid composition (%) of phospholipids in whole cells (B) and the MV fraction (C) was calculated as the amount of each fatty acid divided by the total amount of palmitate, palmitoleate, stearate, oleate, elaidate, linoleate, and linolenate. The results are presented as the mean ± S.D., n = 8 independent experiments. The significant differences among the groups were calculated by a one-way ANOVA followed by the Tukey–Kramer test. (D) The relationship between the fatty acid composition of phospholipids in whole cells and their MV fraction (n = 8).

Analysis of Fatty Acid Composition of Phospholipids in Whole Cells, MVs, and the Plasma Membrane following TNF-α Stimulation

Previously, we reported that stimulation of adipocytes with TNF-α may alter the quality of the MVs.⁵⁾ Therefore, we examined the fatty acid composition of MVs under TNF-α stimulation (Fig. 2A).

Fig. 2. Analysis of Fatty Acid Composition of Phospholipids in Whole Cells and the MV Fraction during the TNF-α-Stimulated State

(A) Schematic representation of stimulation of elaidate-exposed cells with TNF-α. (B) Effect of persistent exposure to elaidate on MV release during the basal or TNF-α-stimulated state (left panel) and TNF-α-dependent MV release (right panel). The results are presented as the mean ± S.D., n = 3 independent experiments. (C, D) Fatty acid composition (%) of phospholipids in whole cells. The MV fraction was calculated as described in Fig. 1B. The results are presented as the mean ± S.D., n = 7 independent experiments. The significant differences among the groups were calculated by one-way ANOVA followed by the Tukey–Kramer test (B–D). (E) Relationship of the fatty acid composition of phospholipids in whole cells and their MV fraction (n = 7).

Although the basal MV release was reduced by exposure to elaidate (Fig. 2B, left panel), MV release induced by TNF-α stimulation was not affected by elaidate treatment (Fig. 2B, right panel). Under these conditions, exposure to elaidate altered the fatty acid composition of phospholipids in whole cells, and the change observed under TNF-α stimulation was similar to that under basal conditions (Fig. 2C). However, the ratio of elaidate in the MV fraction increased in a concentration-dependent manner (Fig. 2D), and the ratios of several fatty acids, including elaidate, in the MV fraction were correlated with those in whole cells (Fig. 2E). Moreover, the ratios of most fatty acids in the phospholipids of whole cells tended to correlate with those in the plasma membrane (Fig. 3). This suggests that treatment with TNF-α alters the quality of the MVs, and alterations in the fatty acid composition of plasma membrane phospholipids are reflected in that of the MVs.

Fig. 3. Relationship between the Fatty Acid Composition of Phospholipids in Whole Cells and Those in the Plasma Membrane during the TNF-α-Stimulated State

Relationship between the fatty acid composition of phospholipids in whole cells and in the plasma membrane (n = 4 independent experiments).

DISCUSSION

In the present study, persistent exposure to elaidate altered the fatty acid composition of phospholipids in the plasma membrane, and these changes were reflected in the MV fraction following TNF-α stimulation; however, the fatty acid composition of whole cells was not correlated with that of the MV fraction during the basal state.

In the basal state, MVs are not uniformly released from the cell membrane but are shed from plasma membrane protrusions.¹²⁾ On the other hand, TNF-α stimulation induces the release of MVs through the lipid raft region.^13,14) Elaidate is likely to be incorporated into phospholipids in the lipid raft (data not shown), resulting in the alteration of the fatty acid composition of phospholipids in whole cells being reflected in MVs only after TNF-α stimulation. On the other hand, little elaidate may be incorporated into the plasma membrane protrusions. Since the fatty acid composition of phospholipids in MVs was clearly different from that in whole cells (Figs. 1 and 2), MVs may be released from specific regions of the plasma membrane. Proteomics and lipidomics of the MVs may reveal the region of MV release and its underlying mechanism.

Endothelial MVs have the potential to monitor fatty acid composition in plasma membrane phospholipids. However, it is necessary to develop isolation methods for endothelial MVs and examine the relationship between the phospholipid fatty acid compositions of whole cells and MVs under various stimuli before in vivo experiments. Because the phospholipid fatty acid composition of endothelial cells is altered under pathological conditions,^15,16) MVs will serve as a useful tool for assessing fatty acid alterations in plasma membrane phospholipids in vivo and the development of disease.

Acknowledgments

This work was supported in part by a Grant-in-Aid for Young Scientists (Grant No. 19K15775 to KI), Scientific Research (C) (Grant Nos. 21K05427 and 23K10944 to KI and GA) from the Japan Society for the Promotion of Science, and ACRO Incubation Grants from Teikyo University.

DECLARATIONS

Author Contributions

KI and GA designed the experiments and conceived the idea for the manuscript. KI, YT, RK, and RO collected and analyzed the data. MS supported data collection and preparation of the manuscript. All authors reviewed the results and approved the final version of the manuscript.

Conflict of Interest

The authors declare no conflict of interest.

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