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Research Article| Volume 283, P19-27, April 2019

Inflammatory adipocyte-derived extracellular vesicles promote leukocyte attachment to vascular endothelial cells

      Highlights

      • Extracellular vesicles (EVs) from inflammed adipocytes increase VCAM expression in vascular endothelial cells.
      • EVs from inflammed and hypoxic adipocytes increase leukocyte attachment to vascular endothelial cells.
      • Inflammation and hypoxia affect the expression of adipokines in adipocytes and EVs.
      • Inflammation and hypoxia affect adipocyte EV yield and size.

      Abstract

      Background and aims

      Obesity is associated with an increased risk of cardiovascular disease, but the mechanisms involved are not completely understood. In obesity, the adipocyte microenvironment is characterised by both hypoxia and inflammation. Therefore, we sought to determine whether extracellular vesicles (EVs) derived from adipocytes in this setting might be involved in mediating cardiovascular disease, specifically by promoting leukocyte attachment to vascular endothelial cells.

      Methods

      Mature 3T3-L1 adipocytes were incubated for 24 h under control, TNF-α (30 ng/mL), hypoxia (1% O2), or TNF-α+hypoxia (30 ng/mL, 1% O2) conditions. EVs were isolated by differential ultracentrifugation and analysed by nanoparticle tracking analysis. Primary human umbilical vein endothelial cells (HUVECs) were treated with EVs for 6 h before being lysed for Western blotting to investigate changes in adhesion molecule production, or for use in leukocyte attachment assays.

      Results

      EVs from adipocytes treated with TNF-α and TNF-α+hypoxia increased vascular cell adhesion molecule (VCAM-1) production in HUVECs compared to basal level (4.2 ± 0.6 and 3.8 ± 0.3-fold increase, respectively (p < 0.05)), an effect that was inhibited by an anti-TNF-α neutralising antibody. Production of other adhesion molecules (E-selectin, P-selectin, platelet endothelial cell adhesion molecule and VE-Cadherin) was unchanged. Pre-incubating HUVECs with TNF-α+hypoxia EVs significantly increased leukocyte attachment compared to basal level (3.0 ± 0.4-fold increase (p < 0.05)).

      Conclusions

      Inflammatory adipocyte EVs induce VCAM-1 production in vascular endothelial cells, accompanied by enhanced leukocyte attachment. Preventing adipocyte derived EV-induced VCAM-1 upregulation may offer a novel therapeutic target in the prevention of obesity-driven cardiovascular disease.

      Graphical abstract

      Keywords

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      References

        • Hubert H.B.
        • Feinleib M.
        • McNamara P.M.
        • Castelli W.P.
        Obesity as an independent risk factor for cardiovascular disease: a 26-year follow-up of participants in the framingham heart study.
        Circulation. 1983; 67: 968-977
        • Ortega F.B.
        • Lee D.C.
        • Katzmarzyk P.T.
        • Ruiz J.R.
        • Sui X.
        • Church T.S.
        • Blair S.N.
        The intriguing metabolically healthy but obese phenotype: cardiovascular prognosis and role of fitness.
        Eur. Heart J. 2013; 34: 389-397
        • Kaur J.
        A comprehensive review on metabolic syndrome.
        Cardiol. Res. Pract. 2014; 2014: 943162
        • Ye J.
        • Gao Z.
        • Yin J.
        • He Q.
        Hypoxia is a potential risk factor for chronic inflammation and adiponectin reduction in adipose tissue of ob/ob and dietary obese mice.
        Am. J. Physiol. Endocrinol. Metab. 2007; 293: E1118-E1128
        • Ye J.
        Emerging role of adipose tissue hypoxia in obesity and insulin resistance.
        Int. J. Obes. 2009; 33: 54-66
        • Pasarica M.
        • Rood J.
        • Ravussin E.
        • Schwarz J.M.
        • Smith S.R.
        • Redman L.M.
        Reduced oxygenation in human obese adipose tissue is associated with impaired insulin suppression of lipolysis.
        J. Clin. Endocrinol. Metab. 2010; 95: 4052-4055
        • Hosogai N.
        • Fukuhara A.
        • Oshima K.
        • Miyata Y.
        • Tanaka S.
        • Segawa K.
        • Furukawa S.
        • Tochino Y.
        • Komuro R.
        • Matsuda M.
        • Shimomura I.
        Adipose tissue hypoxia in obesity and its impact on adipocytokine dysregulation.
        Diabetes. 2007; 56: 901-911
        • Trayhurn P.
        • Wang B.
        • Wood I.S.
        Hypoxia and the endocrine and signalling role of white adipose tissue.
        Arch. Physiol. Biochem. 2008; 114: 267-276
        • Ouchi N.
        • Parker J.L.
        • Lugus J.J.
        • Walsh K.
        Adipokines in inflammation and metabolic disease.
        Nat. Rev. Immunol. 2011; 11: 85-97
        • Bulló M.
        • Salas-Salvadó J.
        • García-Lorda P.
        Adiponectin expression and adipose tissue lipolytic activity in lean and obese women.
        Obes. Surg. 2005; 15: 382-386
        • Ouchi N.
        • Kihara S.
        • Arita Y.
        • Maeda K.
        • Kuriyama H.
        • Okamoto Y.
        • Hotta K.
        • Nishida M.
        • Takahashi M.
        • Nakamura T.
        • Yamashita S.
        • Funahashi T.
        • Matsuzawa Y.
        Novel modulator for endothelial adhesion molecules: adipocyte-derived plasma protein adiponectin.
        Circulation. 1999; 100: 2473-2476
        • Ouedraogo R.
        • Gong Y.
        • Berzins B.
        • Wu X.
        • Mahadev K.
        • Hough K.
        • Chan L.
        • Goldstein B.J.
        • Scalia R.
        Adiponectin deficiency increases leukocyte-endothelium interactions via upregulation of endothelial cell adhesion molecules in vivo.
        J. Clin. Invest. 2007; 117: 1718-1726
        • Bakhai A.
        Adipokines--targeting a root cause of cardiometabolic risk.
        QJM. 2008; 101: 767-776
        • Robbins P.D.
        • Morelli A.E.
        Regulation of immune responses by extracellular vesicles.
        Nat. Rev. Immunol. 2014; 14: 195-208
        • Eguchi A.
        • Lazic M.
        • Armando A.M.
        • Phillips S.A.
        • Katebian R.
        • Maraka S.
        • Quehenberger O.
        • Sears D.D.
        • Feldstein A.E.
        Circulating adipocyte-derived extracellular vesicles are novel markers of metabolic stress.
        J. Mol. Med. (Berl.). 2016; 94: 1241-1253
        • Connolly K.D.
        • Guschina I.A.
        • Yeung V.
        • Clayton A.
        • Draman M.S.
        • Von Ruhland C.
        • Ludgate M.
        • James P.E.
        • Rees D.A.
        Characterisation of adipocyte-derived extracellular vesicles released pre- and post-adipogenesis.
        J. Extracell. Vesicles. 2015; 4: 29159
        • Aoki N.
        • Jin-no S.
        • Nakagawa Y.
        • Asai N.
        • Arakawa E.
        • Tamura N.
        • Tamura T.
        • Matsuda T.
        Identification and characterization of microvesicles secreted by 3t3-l1 adipocytes: redox- and hormone-dependent induction of milk fat globule-epidermal growth factor 8-associated microvesicles.
        Endocrinology. 2007; 148: 3850-3862
        • Kralisch S.
        • Ebert T.
        • Lossner U.
        • Jessnitzer B.
        • Stumvoll M.
        • Fasshauer M.
        Adipocyte fatty acid-binding protein is released from adipocytes by a non-conventional mechanism.
        Int. J. Obes. 2014; 38: 1251-1254
        • Kranendonk M.E.
        • Visseren F.L.
        • van Balkom B.W.
        • Nolte-'t Hoen E.N.
        • van Herwaarden J.A.
        • de Jager W.
        • Schipper H.S.
        • Brenkman A.B.
        • Verhaar M.C.
        • Wauben M.H.
        • Kalkhoven E.
        Human adipocyte extracellular vesicles in reciprocal signaling between adipocytes and macrophages.
        Obesity. 2014; 22: 1296-1308
        • Lever R.
        • Rose M.J.
        • McKenzie E.A.
        • Page C.P.
        Heparanase induces inflammatory cell recruitment in vivo by promoting adhesion to vascular endothelium.
        Am. J. Physiol. Cell Physiol. 2014; 306: C1184-C1190
        • Hagita S.
        • Osaka M.
        • Shimokado K.
        • Yoshida M.
        Adipose inflammation initiates recruitment of leukocytes to mouse femoral artery: role of adipo-vascular axis in chronic inflammation.
        PLoS One. 2011; 6 (e19871)
        • Weisberg S.P.
        • McCann D.
        • Desai M.
        • Rosenbaum M.
        • Leibel R.L.
        • Ferrante A.W.
        Obesity is associated with macrophage accumulation in adipose tissue.
        J. Clin. Invest. 2003; 112: 1796-1808
        • Hotamisligil G.S.
        • Shargill N.S.
        • Spiegelman B.M.
        Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance.
        Science. 1993; 259: 87-91
        • Zhang H.
        • Zhang J.
        • Ungvari Z.
        • Zhang C.
        Resveratrol improves endothelial function: role of tnf{alpha} and vascular oxidative stress.
        Arterioscler. Thromb. Vasc. Biol. 2009; 29: 1164-1171
        • Zhou Z.
        • Connell M.C.
        • MacEwan J.D.
        TNFR1-induced NF-κB, but not ERK, p38MAPK or JNK activation, mediates TNF-induced ICAM-1 and VCAM-1 expression on endothelial cells.
        Cell. Signal. 2007; 19: 1238-1248
        • Granger D.
        • Senchenkova E.
        Inflammation and the Microcirculation.
        1 edition. Morgan & Claypool Life Sciences, San Rafael, California2010
        • Robinson L.A.
        • Tu L.
        • Steeber D.A.
        • Preis O.
        • Platt J.L.
        • Tedder T.F.
        The role of adhesion molecules in human leukocyte attachment to porcine vascular endothelium: implications for xenotransplantation.
        J. Immunol. 1990; 161: 6931-6938
        • Onat D.
        • Brillon D.
        • Colombo P.C.
        • Schmidt A.M.
        Human vascular endothelial cells: a model system for studying vascular inflammation in diabetes and atherosclerosis.
        Curr. Diabetes Rep. 2011; 11: 193-202
        • Cybulsky M.I.
        • Iiyama K.
        • Li H.
        • Zhu S.
        • Chen M.
        • Iiyama M.
        • Davis V.
        • Gutierrez-Ramos J.C.
        • Connelly P.W.
        • Milstone D.S.
        A major role for VCAM-1, but not ICAM-1, in early atherosclerosis.
        J. Clin. Invest. 2001; 107: 1255-1262
        • Durcin M.
        • Fleury A.
        • Taillebois E.
        • Hilairet G.
        • Krupova Z.
        • Henry C.
        • Truchet S.
        • Trötzmüller M.
        • Köfeler H.
        • Mabilleau G.
        • Hue O.
        • Andriantsitohaina R.
        • Martin P.
        • Le Lay S.
        Characterisation of adipocyte-derived extracellular vesicle subtypes identifies distinct protein and lipid signatures for large and small extracellular vesicles.
        J. Extracell. Vesicles. 2017; 6: 1305677
        • Gao C.
        • Boylan B.
        • Fang J.
        • Wilcox D.A.
        • Newman D.K.
        • Newman P.J.
        Heparin promotes platelet responsiveness by potentiating αIIbβ3-mediated outside-in signaling.
        Blood. 2011; 117: 4946-4952
        • Falati S.
        • Liu Q.
        • Gross P.
        • Merrill-Skoloff G.
        • Chou J.
        • Vandendries E.
        • Celi A.
        • Croce K.
        • Furie B.C.
        • Furie B.
        Accumulation of tissue factor into developing thrombi in vivo is dependent upon microparticle P-selectin glycoprotein ligand 1 and platelet P-selectin.
        JEM. 2003; 197: 1585-1598
        • Furuhashi M.
        • Hotamisligil G.S.
        Fatty acid-binding proteins: role in metabolic diseases and potential as drug targets.
        Nat. Rev. Drug Discov. 2008; 7: 489-503
        • Xu A.
        • Wang Y.
        • Xu J.Y.
        • Stejskal D.
        • Tam S.
        • Zhang J.
        • Wat N.M.
        • Wong W.K.
        • Lam K.S.
        Adipocyte fatty acid-binding protein is a plasma biomarker closely associated with obesity and metabolic syndrome.
        Clin. Chem. 2006; 52: 405-413
        • Aragonès G.
        • Saavedra P.
        • Heras M.
        • Cabré A.
        • Girona J.
        • Masana L.
        Fatty acid-binding protein 4 impairs the insulin-dependent nitric oxide pathway in vascular endothelial cells.
        Cardiovasc. Diabetol. 2012; 11: 72
        • Wu L.E.
        • Samocha-Bonet D.
        • Whitworth P.T.
        • Fazakerley D.J.
        • Turner N.
        • Biden T.J.
        • James D.E.
        • Cantley J.
        Identification of fatty acid binding protein 4 as an adipokine that regulates insulin secretion during obesity.
        Mol. Metab. 2014; 3: 465-473
        • Makki K.
        • Froguel P.
        • Wolowczuk I.
        Adipose tissue in obesity-related inflammation and insulin resistance: cells, cytokines, and chemokines.
        ISRN Inflamm. 2013; 2013: 139239
        • Arita Y.
        • Kihara S.
        • Ouchi N.
        • Takahashi M.
        • Maeda K.
        • Miyagawa J.
        • Hotta K.
        • Shimomura I.
        • Nakamura T.
        • Miyaoka K.
        • Kuriyama H.
        • Nishida M.
        • Yamashita S.
        • Okubo K.
        • Matsubara K.
        • Muraguchi M.
        • Ohmoto Y.
        • Funahashi T.
        • Matsuzawa Y.
        Paradoxical decrease of an adipose-specific protein, adiponectin, in obesity.
        Biochem. Biophys. Res. Commun. 1999; 257: 79-83
        • Aprahamian T.R.
        • Sam F.
        Adiponectin in cardiovascular inflammation and obesity.
        Int. J. Inflamm. 2011; 2011: 376909
        • Chen B.
        • Lam K.S.
        • Wang Y.
        • Wu D.
        • Lam M.C.
        • Shen J.
        • Wong L.
        • Hoo R.L.
        • Zhang J.
        • Xu A.
        Hypoxia dysregulates the production of adiponectin and plasminogen activator inhibitor-1 independent of reactive oxygen species in adipocytes.
        Biochem. Biophys. Res. Commun. 2006; 341: 549-556
        • Ivanova E.A.
        • Parolari A.
        • Myasoedova V.
        • Melnichenko A.A.
        • Bobryshev Y.V.
        • Orekhov A.N.
        Peroxisome proliferator-activated receptor (ppar) gamma in cardiovascular disorders and cardiovascular surgery.
        J. Cardiol. 2015; 66: 271-278
        • Yun Z.
        • Maecker H.L.
        • Johnson R.S.
        • Giaccia A.J.
        Inhibition of ppar gamma 2 gene expression by the hif-1-regulated gene dec1/stra13: a mechanism for regulation of adipogenesis by hypoxia.
        Dev. Cell. 2002; 2: 331-341
        • Kern P.A.
        • Di Gregorio G.
        • Lu T.
        • Rassouli N.
        • Ranganathan G.
        Perilipin expression in human adipose tissue is elevated with obesity.
        J. Clin. Endocrinol. Metab. 2004; 89: 1352-1358
        • Wang Y.
        • Sullivan S.
        • Trujillo M.
        • Lee M.J.
        • Schneider S.H.
        • Brolin R.E.
        • Kang Y.H.
        • Werber Y.
        • Greenberg A.S.
        • Fried S.K.
        Perilipin expression in human adipose tissues: effects of severe obesity, gender, and depot.
        Obes. Res. 2003; 11: 930-936
        • Gardiner C.
        • Shaw M.
        • Hole P.
        • Smith J.
        • Tannetta D.
        • Redman C.W.
        • Sargent I.L.
        Measurement of refractive index by nanoparticle tracking analysis reveals heterogeneity in extracellular vesicles.
        J. Extracell. Vesicles. 2014; 24: 25361
        • van der Pol E.
        • Coumans F.A.W.
        • Grootemaat A.E.
        • Gardiner C.
        • Sargent I.L.
        • Harrison P.
        • Sturk A.
        • van Leeuwen T.G.
        • Nieuwland R.
        Particle size distribution of exosomes and microvesicles determined by transmission electron microscopy, flow cytometry, nanoparticle tracking analysis, and resistive pulse sensing.
        JTH. 2014; 12: 1182-1192