Extracellular Vesicles Derived from the Serum of Seriola quinqueradiata Induce Human Umbilical Vein Endothelial Cell Proliferation through Angiogenic Signaling

Abstract

Daily intake of extracellular vesicles (EVs) derived from fish (f-EVs) may contribute to health maintenance by reducing cardiovascular risk. However, their physicochemical and biological properties remain unclear. In this study, we compared the physical characteristics (size, zeta potential, and free fatty acid composition) and biological characteristics (cell proliferation) of f-EVs with those of EVs derived from mammals (m-EVs). In the physical characteristic analysis, f-EVs derived from Pagrus major (PMS-EVs) and Seriola quinqueradiata (SQS-EVs) had a negatively charged and a positively charged group and higher levels of unsaturated fatty acids, unlike m-EVs. In the biological characteristic analysis for f-EVs, SQS-EV enhanced the human umbilical vein endothelial cell proliferation via vascular endothelial growth factor receptor 2, fibroblast growth factor receptor 1, or platelet-derived growth factor β. These data suggest that SQS-EVs have unique functions compared with other EVs. To the best of our knowledge, this is the first study to show that SQS-EVs act positively on human cells.

INTRODUCTION

Fish contain numerous nutrients that are beneficial for human health. Especially, long-chain-omega-3 polyunsaturated fatty acids (ω3-PUFAs) have been shown to reduce cardiovascular risk.¹⁾ However, this reduction in cardiovascular risk cannot be fully achieved by consuming docosahexaenoic acid (DHA)/eicosapentaenoic acid (EPA) supplements alone.²⁾ Therefore, it is crucial to identify fish nutrients other than DHA/EPA that can reduce cardiovascular risk.

In this respect, extracellular vesicle (EVs)-encapsulated functional molecules, such as microRNAs (miRNAs) and proteins secreted from various cells, are a topic of interest in life sciences.³⁾ EVs are attracting attention as new carriers for cell–cell communication.⁴⁾ For example, EVs derived from food, such as cow’s milk, circulate throughout the body. Furthermore, miR-29b, which is present in EVs derived from cow’s milk, regulates the expression of runt-related transcription factor 2 in human cells.⁵⁾ Thus, daily intake of EVs derived from fish (f-EVs) may contribute to the maintenance of health by reducing cardiovascular risk. However, the physiochemical and biological properties of f-EVs are unclear.

In this study, we analyzed the particle size, zeta potential, and free fatty acid composition as fundamental physiochemical properties of EVs derived from fish serum, such as Pagrus major (PMS-EVs) and Seriola quinqueradiata (SQS-EVs), and compared them to EVs derived from mammalian serum, such as fetal bovine serum (FBS-EVs) and human serum (HS-EVs). Moreover, we analyzed the proliferation of human umbilical vein endothelial cells (HUVECs) as a fundamental biological property of f-EVs.

MATERIALS AND METHODS

Preparation of EVs

EVs derived from FBS (Biosera, Couëron, France), HS (HS, Kohjin Bio, Saitama, Japan), Pagrus major serum (PMS, Dainichi, Wakayama, Japan) and Seriola quinqueradiata serum (SQS, Dainichi) were isolated from serum-free conditioned media using the MagCapture™ Exosome Isolation Kit PS (FUJIFILM Wako, Osaka, Japan).

Measurement of Particle Diameters and Zeta Potential

Particle size and zeta potential were measured by dynamic light scattering using a Zetasizer Nano (Malvern, Malvern, U.K.).

Analysis of Free Fatty Acids (FFAs) Derived from EVs

Ice-cold chloroform/methanol (2 : 1, v/v) was added to the EVs purified from 300 µL serum. Subsequently, the chloroform phases were evaporated at room temperature using nitrogen gas, and the dry residue was dissolved in 100 µL of methanol for LC-electrospray ionization (ESI)-MS/MS. HPLC was performed using an ABSCIEX EXION LC AD column (AB SCIEX, MA, U.S.A.) equipped with a CAPCELL PAK UG120 column: 2.0 mm i.d. × 150 mm; OSAKA SODA, Osaka, Japan). A QTRAP 5500 (AB SCIEX) was used to perform quantitation. Other LC-tandem mass spectrometry (LC-MS/MS) conditions were followed in our previous study.⁶⁾

Cell Proliferation Assay

HUVECs (TaKaRa Bio, Shiga, Japan) were maintained at 37 °C in a humidified atmosphere of 5% CO₂ in an endothelial cell growth medium 2 kit (PromoCell, Heidelberg, Germany). HUVECs were cultured in collagen-coated 96-well microplates (5 × 10⁴ cells/well) at 37 °C for 24 h. EVs (5–20 ng of protein) and 64 nM SU5402 (FUJIFILM Wako) were added to each well. The plates were incubated for 24 h at 37 °C in a humidified atmosphere containing 5% CO₂. Cell viability was measured using Cell Count Reagent SF (Nacalai Tesque, Kyoto, Japan) containing WST-8. Absorbance was measured using a Synergy H1 microplate reader (BioTek, VT, U.S.A.) at test and reference wavelengths of 450 and 650 nm, respectively.

Statistical Analysis

All data are expressed as the mean ± standard error of the mean. The data in Fig. 1A were analyzed using Student’s t-test. For other data, differences were compared using a one-way ANOVA followed by Tukey’s test using GraphPad Prism 10. In these analyses, p < 0.05 is considered statistically significant. These p-values were indicated in each figure. In the only cases with p-values under 0.0001, the p-values were indicated as p < 0.0001.

Fig. 1. Free Fatty Acid Compositions of f-EVs

FFA compositions of EVs were semi-quantitatively analyzed using LC-MS/MS. The data were categorized into A) fish and mammalian data (f-EVs: PMS-EVs and SQS-EVs; m-EVs: FBS-EVs and HS-EVs) or B) individual data. Data are shown as the mean and standard deviation (A: n = 6, B: n = 3). p < 0.05 indicates statistical significance and was calculated using the A) Student’s t-test or B) one-way ANOVA followed by Tukey’s test.

RESULTS AND DISCUSSION

Physicochemical Characteristic Analysis of EVs Derived from Fish Compared to EVs Derived from Mammals

To understand the physicochemical characteristics of EVs derived from fish, the particle size, zeta potential, and FFAs composition of PMS-EVs and SQS-EVs as f-EVs were compared with those of FBS-EVs and HS-EVs as m-EVs. The sizes of FBS-EVs and HS-EVs were 128.8 ± 16.7 and 244.5 ± 53.8 nm, respectively, and were comparable to commonly reported EV sizes (approximately 100–300 nm) (Table 1). Under this condition, the sizes of PMS-EVs and SQS-EVs were 199.6 ± 22.4 and 194.1 ± 52.0 nm, respectively). These data suggested that the size of f-EVs was comparable to that of m-EVs.

Table 1. The Particle Size and Zeta Potential of EVs

EVs	Size (nm)	Zeta negative average (mV)	Zeta positive average (mV)
FBS-EVs	128.8 ± 16.7	−22.0 ± 1.9	0
HS-EVs	244.5 ± 53.8	−45.5 ± 11.9	0
PMS-EVs	199.6 ± 22.4	−68.8 ± 11.1	43.6 ± 19.9
SQS-EVs	194.1 ± 52.0	−81.0 ± 13.6	37.2 ± 16.1

Zeta potential analysis of EVs showed that FBS-EVs and HS-EVs had a single negatively charged group. The charge average of FBS-EVs and HS-EVs was −22.0 ± 1.9 and −45.5 ± 11.9 mV, respectively. In contrast, PMS-EVs and SQS-EVs had not only negatively charged groups but also positively charged groups. The negative charge average and positive charge average were −68.8 ± 11.1 and 43.6 ± 19.9 mV in PMS-EVs, and −81.0 ± 13.6 and 37.2 ± 16.1 mV in SQS-EVs, respectively. Few positively charged biomolecules have been reported in small numbers. Dash et al. showed that exosome-derived MCF-7 breast cancer cells have negatively charged and positively charged groups.⁷⁾ However, the physiological functions of exosomes containing positively charged EVs are not understood. Positively charged EVs can easily electrostatically bind to negatively charged biomolecules, such as cationic liposomes (PMID:31641141:2019). Positively charged EVs may play a different role from negatively charged EVs.

Finally, FFA composition of each EV was analyzed. FFAs are a key molecule for homeostasis, because FFAs, especially long-chain polyunsaturated FAs, are a substrate for peroxisome proliferator activated receptor alpha (PPARα) that lowers triglycerides in the beta-oxidation pathway.⁸⁾ Tsutsumi et al. have recently shown that unique long-chain monounsaturated fatty acids (LCMUFAs) in fish oil, such as C20:1 and C22:1, attenuate atherosclerosis development in ApoE −/− and LDLR −/− mice.⁹⁾ In addition, the number of double bonds in FFA defines the flexibility of the lipid membrane because FFAs constitute a lipid bilayer.¹⁰⁾ Thus, it is important to understand the length and degree of FFA unsaturation in analyzing the physicochemical characteristics of f-EVs.

Expression profile analysis of the major FFA species (C16:0, C18:0, C18:1, C18:2, C20:1, C20:4, C20:5, C22:1, and C22:6) showed that the levels of unsaturated FFAs (C18:1, C18:2, C20:1, C20:5, C22:1, and C22:6) in f-EVs were higher than those in m-EVs (Fig. 1A). Moreover, the levels of LCMUFAs (C20:1 and C22:1) and EPA (C20:5) were the highest in PMS-EVs. In particular, the DHA level with C22:6 in both PMS-EVs and SQS-EVs was approximately five-fold higher than that in m-EVs (Fig. 1B). In contrast, saturated FFA (C16:0 and C18:0) levels did not differ significantly between HS-EVs and f-EVs, although FFA levels with C16:0 and C18:0 were lower in FBS-EVs (Figs. 1A, B). These data suggested that f-EVs are softer and more functionally active than m-EVs.

Taken together, f-EVs have unique physicochemical characteristics compared with m-EVs, although the details need to be analyzed in the future. Therefore, f-EVs may have a unique function compared to m-EVs.

Biological Characteristic Analysis of f-EVs Compared to m-EVs

Blood vessels play a central role in cardiovascular events. Therefore, the effects of f-EVs on vascular endothelial cells were analyzed to understand the biological characteristics of f-EVs. HUVEC proliferation analysis revealed that FBS-EVs and HS-EVs inhibited cell growth by more than 20%. Conversely, SQS-EVs enhanced cell growth by >70%, whereas PMS-EVs did not change cell growth (Fig. 2A). These data suggested that SQS-EVs have a unique function in angiogenesis.

Fig. 2. Neutralization of the Angiogenic Signaling-Mediated Cell Growth on EVs by SU5402

HUVECs were treated with A) only EVs (FBS-EVs/ HS-EVs/ PMS-EVs/ SQS-EVs) or B) SQS-EVs and SU5402 for 24 h. HUVEC proliferation was determined by WST-8 assay. Data are shown as the means and standard deviations (n = 4). A) * p < 0.05 (vs. non-treat group; one-way ANOVA followed by Tukey’s test). B) * p < 0.05 (one-way ANOVA followed by Tukey’s test).

To elucidate the mechanism, the angiogenic signal was blocked using SU5402, a potent antagonist of vascular endothelial growth factor receptor 2 (VEGFR2), fibroblast growth factor receptor 1 (FGFR1), and platelet-derived growth factor receptor β (PDGFRβ), which are key receptors for vascular endothelial cell growth. The proliferation assay revealed that SQS-EV treatment reproducibly enhanced cell growth. Under these conditions, cotreatment with SQS-EVs and SU5402 significantly inhibited cell growth (Fig. 2B). The data suggested that SQS-EV could enhance HUVEC proliferation via VEGFR2, FGFR1, or PDGFRβ.

FBS-EVs and HS-EVs inhibited the proliferation of HUVECs. We previously reported that EVs derived from lung epithelial cells suppress tube formation in HUVECs.¹¹⁾ Therefore, EVs can positively and negatively regulate angiogenesis in host cells. In terms of EVs derived from normal cells, it is reasonable to assume that FBS-EVs, HS-EVs, and EVs derived from lung epithelial cells inhibit HUVEC proliferation.

In this assay, SQS-EVs promoted HUVEC proliferation. Nasution et al. reported that snakehead fish extract increased the levels of VEGF, NO, and VEGFR2 expression in cerebral angiogenesis in ischemic stroke rat models, although it was not specified whether the active body expressing the function was an EV.¹²⁾ Therefore, it is reasonable to conclude that SQS-EVs have the potential to promote HUVEC proliferation. However, HUVEC proliferation was not promoted by PMS-EVs, even though these EVs were derived from the same fish. As EVs derived from the same human have different functions, the function of SQS-EVs could be different from that of PMS-EVs, depending on the amount and type of encapsulated functional molecules, such as miRNAs and proteins.

In conclusion, f-EVs have not only a negatively charged group but also a positively charged group and higher levels of unsaturated FAs, unlike m-EVs. Furthermore, in the f-EVs, SQS-EV could enhance HUVEC proliferation via VEGFR2, FGFR1, or PDGFRβ. To the best of our knowledge, this is the first study to show that SQS-EVs act positively on human cells.

Acknowledgments

Financial support was provided by THE TOYO SUISAN FOUNDATION (Tokyo, Japan). We would like to thank Dainichi Corporation (Wakayama, Japan) for supplying the fish serum.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Mozaffarian D, Lemaitre RN, King IB, Song X, Huang H, Sacks FM, Rimm EB, Wang M, Siscovick DS. Plasma phospholipid long-chain omega-3 fatty acids and total and cause-specific mortality in older adults: a cohort study. Ann. Intern. Med., 158, 515–525 (2013).
• 2) Yokoyama M, Origasa H, Matsuzaki M, Matsuzawa Y, Saito Y, Ishikawa Y, Oikawa S, Sasaki J, Hishida H, Itakura H, Kita T, Kitabatake A, Nakaya N, Sakata T, Shimada K, Shirato K. Effects of eicosapentaenoic acid on major coronary events in hypercholesterolaemic patients (JELIS): a randomised open-label, blinded endpoint analysis. Lancet, 369, 1090–1098 (2007).
• 3) Kosaka N, Iguchi H, Yoshioka Y, Takeshita F, Matsuki Y, Ochiya T. Secretory mechanisms and intercellular transfer of microRNAs in living cells. J. Biol. Chem., 285, 17442–17452 (2010).
• 4) Kalluri R, McAndrews KM. The role of extracellular vesicles in cancer. Cell, 186, 1610–1626 (2023).
• 5) Baier SR, Nguyen C, Xie F, Wood JR, Zempleni J. MicroRNAs are absorbed in biologically meaningful amounts from nutritionally relevant doses of cow milk and affect gene expression in peripheral blood mononuclear cells, HEK-293 kidney cell cultures, and mouse livers. J. Nutr., 144, 1495–1500 (2014).
• 6) Aizawa F, Sato S, Yamazaki F, Yao I, Yamashita T, Nakamoto K, Kasuya F, Setou M, Tokuyama S. N-3 fatty acids modulate repeated stress-evoked pain chronicity. Brain Res., 1714, 218–226 (2019).
• 7) Dash M, Palaniyandi K, Ramalingam S, Sahabudeen S, Raja NS. Exosomes isolated from two different cell lines using three different isolation techniques show variation in physical and molecular characteristics. Biochim. Biophys. Acta Biomembr., 1863, 183490 (2021).
• 8) Hardwick JP, Osei-Hyiaman D, Wiland H, Abdelmegeed MA, Song BJ. PPAR/RXR regulation of fatty acid metabolism and fatty acid omega-hydroxylase (CYP4) isozymes: implications for prevention of lipotoxicity in fatty liver disease. PPAR Res., 2009, 952734 (2009).
• 9) Tsutsumi R, Yamasaki Y, Takeo J, Miyahara H, Sebe M, Bando M, Tanba Y, Mishima Y, Takeji K, Ueshima N, Kuroda M, Masumoto S, Harada N, Fukuda D, Yoshimoto R, Tsutsumi YM, Aihara KI, Sata M, Sakaue H. Long-chain monounsaturated fatty acids improve endothelial function with altering microbial flora. Transl. Res., 237, 16–30 (2021).
• 10) Saeedimasine M, Montanino A, Kleiven S, Villa A. Role of lipid composition on the structural and mechanical features of axonal membranes: a molecular simulation study. Sci. Rep., 9, 8000 (2019).
• 11) Yamashita T, Kamada H, Kanasaki S, Nagano K, Inoue M, Higashisaka K, Yoshioka Y, Tsutsumi Y, Tsunoda S. Ephrin type-A receptor 2 on tumor-derived exosomes enhances angiogenesis through the activation of MAPK signaling. Pharmazie, 74, 614–619 (2019).
• 12) Nasution I, Sjahrir H, Ilyas S, Ichwan M. Snakehead fish extract as an enhancer of vascular endothelial growth factor and nitric oxide levels in cerebral angiogenesis: an insight of stroke therapy. Med. Glas. (Zenica), 17, 420–424 (2020).