Loss of fatty acid binding protein 3 ameliorates lipopolysaccharide-induced inflammation and endothelial dysfunction
2023; Elsevier BV; Volume: 299; Issue: 3 Linguagem: Inglês
10.1016/j.jbc.2023.102921
ISSN1083-351X
AutoresHien C. Nguyen, Shuhan Bu, Sepideh Nikfarjam, Berk Rasheed, David Michels, Aman Singh, Shweta Singh, Caroline Marszal, John J. McGuire, Qingping Feng, Jefferson C. Frisbee, Mohammad Qadura, Krishna K. Singh,
Tópico(s)Inflammatory mediators and NSAID effects
ResumoCirculating fatty acid–binding protein 3 (FABP3) is an effective biomarker of myocardial injury and peripheral artery disease (PAD). The endothelium, which forms the inner most layer of every blood vessel, is exposed to higher levels of FABP3 in PAD or following myocardial injury, but the pathophysiological role of endothelial FABP3, the effect of FABP3 exposure on endothelial cells, and related mechanisms are unknown. Here, we aimed to evaluate the pathophysiological role of endothelial FABP3 and related mechanisms in vitro. Our molecular and functional in vitro analyses show that (1) FABP3 is basally expressed in endothelial cells; (2) inflammatory stress in the form of lipopolysaccharide (LPS) upregulated endothelial FABP3 expression; (3) loss of endogenous FABP3 protected endothelial cells against LPS-induced endothelial dysfunction; however, exogenous FABP3 exposure exacerbated LPS-induced inflammation; (4) loss of endogenous FABP3 protected against LPS-induced endothelial dysfunction by promoting cell survival and anti-inflammatory and pro-angiogenic signaling pathways. Together, these findings suggest that gain-of endothelial FABP3 exacerbates, whereas loss-of endothelial FABP3 inhibits LPS-induced endothelial dysfunction by promoting cell survival and anti-inflammatory and pro-angiogenic signaling. We propose that an increased circulating FABP3 in myocardial injury or PAD patients may be detrimental to endothelial function, and therefore, therapies aimed at inhibiting FABP3 may improve endothelial function in diseased states. Circulating fatty acid–binding protein 3 (FABP3) is an effective biomarker of myocardial injury and peripheral artery disease (PAD). The endothelium, which forms the inner most layer of every blood vessel, is exposed to higher levels of FABP3 in PAD or following myocardial injury, but the pathophysiological role of endothelial FABP3, the effect of FABP3 exposure on endothelial cells, and related mechanisms are unknown. Here, we aimed to evaluate the pathophysiological role of endothelial FABP3 and related mechanisms in vitro. Our molecular and functional in vitro analyses show that (1) FABP3 is basally expressed in endothelial cells; (2) inflammatory stress in the form of lipopolysaccharide (LPS) upregulated endothelial FABP3 expression; (3) loss of endogenous FABP3 protected endothelial cells against LPS-induced endothelial dysfunction; however, exogenous FABP3 exposure exacerbated LPS-induced inflammation; (4) loss of endogenous FABP3 protected against LPS-induced endothelial dysfunction by promoting cell survival and anti-inflammatory and pro-angiogenic signaling pathways. Together, these findings suggest that gain-of endothelial FABP3 exacerbates, whereas loss-of endothelial FABP3 inhibits LPS-induced endothelial dysfunction by promoting cell survival and anti-inflammatory and pro-angiogenic signaling. We propose that an increased circulating FABP3 in myocardial injury or PAD patients may be detrimental to endothelial function, and therefore, therapies aimed at inhibiting FABP3 may improve endothelial function in diseased states. The fatty acid–binding proteins (FABPs) are a family of transport proteins for fatty acids and other lipophilic substances between extracellular and intracellular membranes and receptors and play an important role in the regulation of lipid homeostasis (1Elmasri H. Ghelfi E. Yu C. Traphagen S. Cernadas M. Cao H. et al.Endothelial cell-fatty acid binding protein 4 promotes angiogenesis: role of stem cell factor/c-kit pathway.Angiogenesis. 2012; 15: 457-468Crossref PubMed Scopus (112) Google Scholar). FABPs are also involved in the production of the cell membrane in the endoplasmic reticulum and various enzymatic activities in the cytosol (2Sprecher H. Metabolism of highly unsaturated n-3 and n-6 fatty acids.Biochim. Biophys. 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The myocardial isoform, heart-type fatty acid–binding protein, is encoded by the FABP3 gene. Besides its abundant expression in the cardiomyocytes, FABP3 is also expressed significantly in other cell types (5Tsukahara R. Haniu H. Matsuda Y. Tsukahara T. Heart-type fatty-acid-binding protein (FABP3) is a lysophosphatidic acid-binding protein in human coronary artery endothelial cells.FEBS Open Bio. 2014; 4: 947-951Crossref PubMed Scopus (14) Google Scholar). Their lipid-trafficking mechanism is essential for the metabolic homeostasis of cardiac function (6Stanley W.C. Recchia F.A. Lopaschuk G.D. Myocardial substrate metabolism in the normal and failing heart.Physiol. Rev. 2005; 85: 1093-1129Crossref PubMed Scopus (1466) Google Scholar). For their unique cardiac-expression profile, FABP3 has been proposed as an effective biomarker of myocardial injury (7Glatz J.F. Kleine A.H. van Nieuwenhoven F.A. Hermens W.T. van Dieijen-Visser M.P. van der Vusse G.J. Fatty-acid-binding protein as a plasma marker for the estimation of myocardial infarct size in humans.Br. Heart J. 1994; 71: 135-140Crossref PubMed Google Scholar) as FABP3 is readily released from heart muscles into the blood following a heart attack (8Rezar R. Jirak P. Gschwandtner M. Derler R. Felder T.K. Haslinger M. et al.Heart-type fatty acid-binding protein (H-FABP) and its role as a biomarker in heart failure: what do we know so far?.J. Clin. Med. 2020; 9: E164Crossref PubMed Scopus (34) Google Scholar, 9Bivona G. Agnello L. Bellia C. Lo Sasso B. Ciaccio M. Diagnostic and prognostic value of H-FABP in acute coronary syndrome: still evidence to bring.Clin. Biochem. 2018; 58: 1-4Crossref PubMed Scopus (36) Google Scholar, 10Xu L.-Q. Yang Y.-M. Tong H. Xu C.-F. 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Aside from the general lipid-trafficking mechanism and its feature as a cardiac biomarker, the unique function of FABP3 remains largely unknown, particularly its roles in cardiovascular diseases (CVDs). Systemic infections, or sepsis, have been reported to exacerbate cardiac injuries in atherosclerotic patients (13Madjid M. Vela D. Khalili-Tabrizi H. Casscells S.W. Litovsky S. Systemic infections cause exaggerated local inflammation in atherosclerotic coronary arteries: clues to the triggering effect of acute infections on acute coronary syndromes.Tex. Heart Inst. J. 2007; 34: 11-18PubMed Google Scholar). Physiologically, the body's lipids contribute not only as an efficient source of energy but also as a source of regulatory signals maintaining proper systemic functions or homeostasis, such as hormonal balance (14Bhathena S.J. Relationship between fatty acids and the endocrine and neuroendocrine system.Nutr. 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Accordingly, recently we identified increased circulating levels of FABP3 in peripheral arterial disease (PAD) patients with severe inflammation and particularly undergoing critical limb ischemia, who were negative for any signs of cardiac damage (18Syed M.H. Zamzam A. Khan H. Singh K. Forbes T.L. Rotstein O. et al.Fatty acid binding protein 3 is associated with peripheral arterial disease.JVS Vasc. Sci. 2020; 1: 168-175Abstract Full Text Full Text PDF PubMed Google Scholar). The endothelium lines the inner walls of all blood vessels and is in direct contact with blood and regulates tissue–blood metabolic and signaling exchanges, vascular homeostasis, and inflammation; impaired endothelial function or endothelial dysfunction is a key mechanism behind CVDs (14Bhathena S.J. Relationship between fatty acids and the endocrine and neuroendocrine system.Nutr. Neurosci. 2006; 9: 1-10Crossref PubMed Scopus (37) Google Scholar, 19Pober J.S. Min W. Bradley J.R. 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Biochem. 2018; 58: 1-4Crossref PubMed Scopus (36) Google Scholar, 10Xu L.-Q. Yang Y.-M. Tong H. Xu C.-F. Early diagnostic performance of heart-type fatty acid binding protein in suspected acute myocardial infarction: evidence from a meta-analysis of contemporary studies.Heart Lung Circ. 2018; 27: 503-512Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). However, the source of FABP3 and its effect on the endothelium remains largely unknown, and the role of endothelial FABP3 has not been fully characterized at baseline and under stress conditions. Accordingly, our objective is to evaluate (1) the endothelium as a potential source of FABP3, (2) the role of endothelial FABP3 in endothelial function and survival, and (3) the effect of increased FABP3 exposure on endothelial cell function and inflammation at baseline and after stress and (4) related mechanisms. Chronic inflammation is the central driving mechanism between endothelial dysfunction and CVDs (21Taleb S. Inflammation in atherosclerosis.Arch. Cardiovasc. Dis. 2016; 109: 708-715Crossref PubMed Scopus (217) Google Scholar, 22Libby P. Buring J.E. Badimon L. Hansson G.K. Deanfield J. Bittencourt M.S. et al.Atherosclerosis.Nat. Rev. Dis. Primers. 2019; 5: 56Crossref PubMed Scopus (974) Google Scholar). Inflammation is also a common factor between myocardial ischemia/heart failure (23Riehle C. Bauersachs J. Key inflammatory mechanisms underlying heart failure.Herz. 2019; 44: 96-106Crossref PubMed Scopus (0) Google Scholar) and PADs (24Brevetti G. Giugliano G. Brevetti L. Hiatt W.R. Inflammation in peripheral artery disease.Circulation. 2010; 122: 1862-1875Crossref PubMed Scopus (208) Google Scholar), which are associated with increased circulatory FABP3 and thereby increased FABP3 exposure to endothelial cells. Lipopolysaccharide (LPS), a Gram-negative bacterial endotoxin, is known to induce severe inflammation and endothelial dysfunction (25Dauphinee S.M. Karsan A. Lipopolysaccharide signaling in endothelial cells.Lab. Invest. 2006; 86: 9-22Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar); accordingly, LPS is extensively used in experimental models to study inflammation and associated endothelial dysfunction in vitro and in vivo (25Dauphinee S.M. Karsan A. Lipopolysaccharide signaling in endothelial cells.Lab. Invest. 2006; 86: 9-22Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar, 26Yücel G. Zhao Z. El-Battrawy I. Lan H. Lang S. Li X. et al.Lipopolysaccharides induced inflammatory responses and electrophysiological dysfunctions in human-induced pluripotent stem cell derived cardiomyocytes.Sci. Rep. 2017; 7: 2935Crossref PubMed Scopus (92) Google Scholar, 27Singh K.K. Matkar P.N. Muhammad S. Quan A. Gupta V. Teoh H. et al.Investigation of novel LPS-induced differentially expressed long non-coding RNAs in endothelial cells.Mol. Cell Biochem. 2016; 421: 157-168Crossref PubMed Scopus (21) Google Scholar, 28Cao Y. Gong Y. Liu L. Zhou Y. Fang X. Zhang C. et al.The use of human umbilical vein endothelial cells (HUVECs) as an in vitro model to assess the toxicity of nanoparticles to endothelium: a review.J. Appl. Toxicol. 2017; 37: 1359-1369Crossref PubMed Scopus (178) Google Scholar). Our data demonstrate that endothelial cells basally express FABP3; inflammation, in the form of LPS treatment, significantly upregulates endothelial FABP3 expression. Furthermore, loss-of-endothelial FABP3 inhibits LPS-induced endothelial dysfunction by promoting cell survival and anti-inflammatory and pro-angiogenic pathways. In contrast, gain-of-endothelial FABP3 appears to exacerbate inflammation and endothelial function. Our results suggest that elevated FABP3 in myocardial injury or PAD may be detrimental to the endothelium; therefore, therapies aimed at inhibiting serum FABP3 may improve endothelial function in diseased states. Our FABP3 quantitative polymerase chain reaction (qPCR) data on vehicle-treated (control) endothelial cells confirmed the basal expression of FABP3 in human umbilical vein endothelial cells (HUVECs) (Fig. 1A). Next, to evaluate the effect of inflammation in endothelial cells in the form of LPS-treatment on FABP3 expression, we treated endothelial cells with different doses of LPS (10, 20, 50, 100, and 200 ng/ml) or vehicle control for 24 h and then measured the FABP3 expression. Our qPCR data show significant upregulation of FABP3 in endothelial cells by all the doses of LPS-treatment (Fig. 1A). Maximum but similar FABP3 expression was observed for 100 and 200 ng/ml of LPS, and accordingly, 100 ng/ml was chosen to be the experimental dose to evaluate the effect of loss of FABP3 on LPS-induced endothelial dysfunction. A similar dose has been used by many other comparable studies in endothelial cells (29Anand A.R. Cucchiarini M. Terwilliger E.F. Ganju R.K. The tyrosine kinase Pyk2 mediates lipopolysaccharide-induced IL-8 expression in human endothelial cells.J. Immunol. 2008; 180: 5636-5644Crossref PubMed Scopus (40) Google Scholar, 30Cai G.-L. Yang Z.-X. Guo D.-Y. Hu C.-B. Yan M.-L. Yan J. Macrophages enhance lipopolysaccharide induced apoptosis via Ang1 and NF-κB pathways in human umbilical vein endothelial cells.Sci. Rep. 2021; 11: 2918Crossref PubMed Scopus (2) Google Scholar). We also evaluated the effect of time on LPS-induced FABP3 upregulation and observed that the FABP3 was upregulated as early as 1-h posttreatment (Fig. 1B). We then tested whether LPS-induced FABP3 upregulation is associated with increased secretion of FABP3 in the culture medium and observed increased LPS treatment–induced secretion of FABP3 in the culture medium (Fig. 1C). To understand the effect of LPS-induced upregulation of FABP3 on endothelial function, we successfully silenced FABP3 in HUVECs and observed ∼90% reduction at the transcript level (Fig. 1D). FABP3-silencing was also confirmed at the protein level by Western blotting for FABP3 (Fig. 1E). We then treated FABP3-silenced and control endothelial cells with 100 ng/ml of LPS and evaluated endothelial function in the form of tube-forming, migratory, and proliferative potential of endothelial cells. To our surprise, the loss of FABP3 significantly increased the number of nodes and tube length in FABP3-silenced versus control endothelial cells (Fig. 1, F–H). LPS treatment is known to inhibit tube-forming potential (31Xu F. Zhou F. Inhibition of microRNA-92a ameliorates lipopolysaccharide-induced endothelial barrier dysfunction by targeting ITGA5 through the PI3K/Akt signaling pathway in human pulmonary microvascular endothelial cells.Int. Immunopharmacol. 2020; 78106060Crossref Scopus (20) Google Scholar); accordingly, we also observed significant inhibition of tube formation in LPS-treated scrambled-transfected versus vehicle-treated scrambled-transfected control endothelial cells (Fig. 1, F–H). Interestingly, loss of FABP3 was able to significantly restore tube length in LPS-treated FABP3-deficient in comparison to LPS-treated control endothelial cells (Fig. 1, F and H). However, the loss of FABP3 showed no effect on the LPS-induced inhibition of the number of nodes in HUVECs (Fig. 1, F and G). Next, to understand the effect of LPS treatment on the migratory capacity of FABP3-deficient endothelial cells, we measured migratory capacity via scratch assay (32Liang C.-C. Park A.Y. Guan J.-L. In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro.Nat. Protoc. 2007; 2: 329-333Crossref PubMed Scopus (3141) Google Scholar). Loss of FABP3 and LPS treatment appeared to inhibit and upregulate endothelial cell migration, respectively (Fig. 1, I and J). LPS-induced upregulation of endothelial cell migration has been previously reported depending on specific dosages (33Zheng X. Zhang W. Hu X. Different concentrations of lipopolysaccharide regulate barrier function through the PI3K/Akt signalling pathway in human pulmonary microvascular endothelial cells.Sci. Rep. 2018; 8: 9963Crossref PubMed Scopus (50) Google Scholar); however, loss of FABP3 was able to attenuate LPS's effect on endothelial cell migration (Fig. 1, I and J). We did observe a trend toward increased LPS-induced migration, but the difference was nonsignificant and that can be attributed to the sensitivity of the method used. We then evaluated the effect of loss of FABP3 and LPS on the proliferative capacity of endothelial cells via measuring the cell count using the CytoSmart Automated Cell Counter. Loss of FABP3 appeared not to affect endothelial cell proliferation; however, LPS treatment significantly inhibited the proliferative potential of endothelial cells, which was, interestingly, restored in the FABP3 silenced and LPS treated in comparison to LPS-treated scrambled control-transfected endothelial cells (Fig. 1K). Next, to understand whether LPS-induced reduced cell proliferation is associated with increased cell death and whether the loss of FABP3 is associated with the restoration of cell proliferation is due to increased survival, we measured apoptosis in FABP3-silenced and LPS-treated endothelial cells. Our Western blot data demonstrated the absence of cleaved-CASPASE3 protein in the siFABP3-transfected endothelial cells, suggesting that LPS-induced apoptosis in endothelial cells was inhibited by loss of FABP3 in LPS-treated endothelial cells (Fig. 1L). Overall, these data indicate that loss of FABP3 protects against LPS-induced endothelial dysfunction by restoring angiogenic, migratory, and proliferative potential and by inhibiting LPS-induced apoptosis of endothelial cells. To understand the effect of loss of FABP3 and LPS on the molecular and regulatory level in endothelial cells, we evaluated the expression and activation of the essential regulators of endothelial function. Endothelial nitric oxide synthase (eNOS) and protein kinase B (AKT) are the two essential regulators of endothelial function (34Heiss C. Rodriguez-Mateos A. Kelm M. Central role of eNOS in the maintenance of endothelial homeostasis.Antioxid. Redox Signal. 2015; 22: 1230-1242Crossref PubMed Scopus (113) Google Scholar). LPS is known to inhibit eNOS expression and activation (35Piepot H.A. Boer C. Groeneveld A.B. Van Lambalgen A.A. Sipkema P. Lipopolysaccharide impairs endothelial nitric oxide synthesis in rat renal arteries.Kidney Int. 2000; 57: 2502-2510Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), and accordingly, we also observed a reduction in the eNOS protein expression and activation levels in LPS-treated endothelial cells (Fig. 2, A–C). Interestingly, we observed a significantly higher protein level of eNOS in FABP3-silenced endothelial cells, which also corresponded with increased phosphorylation of eNOS (Fig. 2, A–C). LPS-associated inhibition of eNOS expression and activation was restored in LPS-treated FABP3-silenced endothelial cells (Fig. 2, A–C). Given that the phosphatidylinositol 3-kinase (PI3K)/AKT/eNOS signaling pathway is critical for the maintenance of endothelial function and that activated AKT can directly activate eNOS (34Heiss C. Rodriguez-Mateos A. Kelm M. Central role of eNOS in the maintenance of endothelial homeostasis.Antioxid. Redox Signal. 2015; 22: 1230-1242Crossref PubMed Scopus (113) Google Scholar), we next measured total and activated AKT levels in FABP3-silenced and LPS-treated endothelial cells. LPS has been shown to compromise AKT activation (36Wu J. Li X. Huang L. Jiang S. Tu F. Zhang X. et al.HSPA12B inhibits lipopolysaccharide-induced inflammatory response in human umbilical vein endothelial cells.J. Cell Mol. Med. 2015; 19: 544-554Crossref PubMed Google Scholar); accordingly, we also observed reduced AKT activation in LPS-treated endothelial cells (Fig. 2D). However, to our surprise, when we quantified and evaluated the activated versus total AKT, the inhibition was not significant between the LPS-treated siFABP3- and scrambles-transfected HUVECs (Fig. 2E). Next, we questioned whether this lack of difference is due to inhibition of total AKT expression by LPS treatment in endothelial cells and quantified total AKT. As expected, LPS significantly inhibited total AKT expression in endothelial cells (Fig. 2F). Interestingly, AKT expression was restored in LPS-treated FABP3-silenced endothelial cells (Fig. 2F), and when we quantified activated AKT (p-AKT), we observed a significant upregulation again for both FABP3-silenced endothelial cells and LPS-treated FABP3-silenced endothelial cells (Fig. 2, D and H). Protein p21, a cell cycle inhibitor, is known to regulate endothelial cell proliferation physiologically and also in pathological conditions (37Mühleder S. Fernández-Chacón M. Garcia-Gonzalez I. Benedito R. Endothelial sprouting, proliferation, or senescence: tipping the balance from physiology to pathology.Cell Mol. Life Sci. 2021; 78: 1329-1354Crossref PubMed Scopus (16) Google Scholar). Most importantly, LPS-mediated inhibition of cell proliferation has been previously attributed to p21 upregulation (38Kaneko Y.S. Ota A. Nakashima A. Nagasaki H. Kodani Y. Mori K. et al.Lipopolysaccharide treatment arrests the cell cycle of BV-2 microglial cells in G₁ phase and protects them from UV light-induced apoptosis.J. Neural Transm. (Vienna). 2015; 122: 187-199Crossref PubMed Scopus (3) Google Scholar). Accordingly, we measured the p21 expression in FABP3-silenced and LPS-treated endothelial cells. Our transcript data showed a significant reduction in p21 transcript level in FABP3-silenced endothelial cells; p21 transcript and protein appeared to be upregulated in LPS-treated endothelial cells, whereas the p21 expression was restored in LPS-treated FABP3-silenced endothelial cells in comparison to LPS-treated scrambled control-transfected endothelial cells (Fig. 2, I–K). These data indicated that loss of FABP3-associated restoration of endothelial function in LPS-treated endothelial cells is mediated by increased AKT/eNOS signaling and inhibition of LPS-associated p21 expression. To assess the role of FABP3 in endothelial inflammation, we evaluated the expression level of key inflammatory markers, including the ICAM-1, VCAM-1, and E-SELECTIN, and the secretory inflammatory cytokines, such as IL1b, IL6, and MCP-1, in FABP3-silenced and LPS-treated endothelial cells. LPS-treatment is known to induce ICAM-1 and VCAM-1 expression (39Sawa Y. Ueki T. Hata M. Iwasawa K. Tsuruga E. Kojima H. et al.LPS-induced IL-6, IL-8, VCAM-1, and ICAM-1 expression in human lymphatic endothelium.J. Histochem. Cytochem. 2008; 56: 97-109Crossref PubMed Scopus (115) Google Scholar); accordingly, we also observed a significant induction of ICAM-1 (Fig. 3, A–C) and VCAM-1 (Fig. 3, D–F) in the LPS-treated scrambled control-transfected endothelial cells. Loss of FABP3 significantly inhibited LPS-induced expression of ICAM-1 at both the transcript and protein levels in HUVECs (Fig. 3, A–C). LPS-induced VCAM-1 transcript level also appeared to be inhibited by loss-of FABP3 in endothelial cells; however, to our surprise, these data did not translate to the protein levels, where we observed further increased level of VCAM-1 in the LPS-treated FABP3-silenced endothelial cells versus LPS-treated scrambled control-transfected endothelial cells (Fig. 3, D–F). Similar to ICAM-1 and VCAM-1, the expression level of E-SELECTIN was induced by LPS, which was again restored by loss of FABP3 in LPS-treated FABP3-silenced endothelial cells (Fig. 3G). LPS is also known to promote the expression of inflammatory cytokines, such as interleukins, IL1b and IL6, and the chemoattractant factor MCP-1 (40Li W. Yang S. Kim S.O. Reid G. Challis J.R.G. Bocking A.D. Lipopolysaccharide-induced profiles of cytokine, chemokine, and growth factors produced by human decidual cells are altered by lactobacillus rhamnosus GR-1 supernatant.Reprod. Sci. 2014; 21: 939-947Crossref PubMed Scopus (37) Google Scholar). Accordingly, we observed LPS-induced significant upregulation in the expression level of IL1b and IL6 along with the expression of MCP-1 in endothelial cells (Fig. 3, H–J). Interestingly, loss of FABP3 was successfully able to significantly inhibit the expression of all these studied inflammatory molecules in LPS-treated FABP3-silenced endothelial cells (Fig. 3, H–J). Taken together, these data indicate that loss of FABP3 protects against LPS-induced inflammation in endothelial cells. Next, to understand the effect of exogenous exposure of FABP3 on endothelial inflammation basally and after LPS-stimulation, we treated endothelial cells with different doses of recombinant human FABP3 (rhFABP3) and LPS and then measured the expression level of ICAM-1 and VCAM-1. Recombinant human FABP3 alone did not significantly affect the inflammation, measured in the form of ICAM-1 and VCAM-1 expression; however, rhFABP3 significantly increased ICAM-1 and VCAM-1 transcripts in LPS-treated endothelial cells, demonstrating an additive effect (Fig. 4, A and B). Given the observed discrepancy between transcript and protein levels in LPS-treated FABP3-deficient endothelial cells, we measured the expression level of ICAM1 and VCAM1 in rhFABP3 and LPS–treated endothelial cells. We observed an expected result, where VCAM-1 and ICAM-1 protein were increased in rhFABP3 and LPS–treated endothelial cells in comparison to LPS only–treated endothelial cells (Fig. 4C). Next, to assess the effect of rhFAB3 exposure on endothelial cell function in vivo, we measured acetylcholine-induced relaxations using myography with isolated aortas from wildtype mice (41Hennessey J.C. McGuire J.J. Attenuated vasodilator effectiveness of protease-activated receptor 2 agonist in heterozygous par2 knockout mice.PLoS One. 2013; 8e55965Crossref Scopus (6) Google Scholar). There appears to be a small effect of increasing relaxation (<10%) of phenylephrine-contracted aortas by acetylcholine in the rhF
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