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International Journal of Molecular Sciences

Article Endothelial Mediates the Anti-Atherosclerotic Action of Torenia concolor Lindley var. Formosama Yamazaki

1, 2, 3 4 5, Li-Ching Cheng †, Bei-Chia Guo †, Chia-Hui Chen , Chi-Jen Chang , Ta-Sen Yeh * and Tzong-Shyuan Lee 2,* 1 Department of Nursing, Division of Basic Medical Sciences, Chang Gung University of Science and Technology, Taoyuan 33303, Taiwan; [email protected] 2 Graduate Institute and Department of Physiology, College of Medicine, National Taiwan University, Taipei 10051, Taiwan; [email protected] 3 Institute and Department of Physiology, School of Medicine, National Yang-Ming University, Taipei 11221, Taiwan; [email protected] 4 The First Cardiovascular Division, Chang Gung Memorial Hospital, Taoyuan 33305, Taiwan; [email protected] 5 Department of General Surgery, Chang Gung Memorial Hospital, Chang Gung University, Taoyuan 33305, Taiwan * Correspondence: [email protected] (T.-S.Y.); [email protected] (T.-S.L.); Tel.: +886-3-3281200 (T.-S.Y.); +886-2-23123456 (T.-S.L.) These authors contributed equally to this work. †  Received: 31 December 2019; Accepted: 17 February 2020; Published: 24 February 2020 

Abstract: Torenia concolor Lindley var. formosama Yamazaki ethanolic extract (TCEE) is reported to have anti-inflammatory and anti-obesity properties. However, the effects of TCEE and its underlying mechanisms in the activation of endothelial (eNOS) have not yet been investigated. Increasing the -derived nitric oxide (NO) production has been known to be beneficial against the development of cardiovascular diseases. In this study, we investigated the effect of TCEE on eNOS activation and NO-related endothelial function and inflammation by using an in vitro system. In endothelial cells (ECs), TCEE increased NO production in a concentration-dependent manner without affecting the expression of eNOS. In addition, TCEE increased the of eNOS at 635 residue (Ser635) and Ser1179, Akt at Ser473, kinase II (CaMKII) at residue 286 (Thr286), and AMP-activated protein kinase (AMPK) at Thr172. Moreover, TCEE-induced NO production, and EC proliferation, migration, and tube formation were diminished by pretreatment with LY294002 (an Akt inhibitor), KN62 (a CaMKII inhibitor), and compound C (an AMPK inhibitor). Additionally, TCEE attenuated the tumor necrosis factor-α-induced inflammatory response as evidenced by the expression of adhesion molecules in ECs and monocyte adhesion onto ECs. These inflammatory effects of TCEE were abolished by L-NG- methyl ester (an NOS inhibitor). Moreover, chronic treatment with TCEE attenuated hyperlipidemia, systemic and aortic inflammatory response, and the atherosclerotic lesions in apolipoprotein E-deficient mice. Collectively, our findings suggest that TCEE may confer protection from by preventing endothelial dysfunction.

Keywords: Torenia concolor Lindley var. formosama Yamazak; endothelial nitric oxide synthase; nitric oxide; anti-inflammatory effect; atherosclerosis

1. Introduction The endothelium is a monolayered continuous cell sheet lining the luminal surface of vessel walls that not only serves as the cross-bridge of communication between the blood and cells but also

Int. J. Mol. Sci. 2020, 21, 1532; doi:10.3390/ijms21041532 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 1532 2 of 17 actively regulates the functions of surrounding cells through complex signaling pathways [1,2]. Under certain circumstances, such as and atherosclerosis, modified LDL impairs the function of the endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) system and then upregulates the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in endothelial cell (ECs), leading to the recruitment of monocytes into the subendothelial space of the vessel wall [3,4]. These events have been considered as one of the earliest pathophysiological manifestations of atherosclerosis [5,6]. Several lines of evidence have clearly indicated the crucial role of endothelium-derived NO, whereby it regulates various physiological functions, including vessel relaxation, proliferation and migration of ECs, inhibition of activation, and attenuation of inflammatory responses in the vessel wall [7]. Impaired NO production has been considered one of the earliest pathophysiological manifestations for endothelial dysfunction and is highly associated with the prevalence of inflammatory diseases and cardiovascular diseases [8–10]. The regulation of eNOS is tightly regulated not only at the transcriptional level but also by post-translational mechanisms [11,12]. It is well known that eNOS can be activated by several physical and chemical stimuli, such as shear stress, estrogen, or bradykinin, and through kinase-dependent signaling pathways, including the PI3K/Akt, calmodulin kinase II (CaMK II), or AMP-activated protein kinase (AMPK) pathways [11,12]. Increasing the activity of eNOS-NO signaling has been considered a therapeutic strategy for the treatment of cardiovascular diseases [13,14]. During the past decade, considerable efforts have been taken to discover the potential of traditional Chinese medicine in many fields, particularly cancers, inflammatory diseases, and cardiovascular diseases [15–20]. Torenia concolor Lindley var. formosana Yamazaki (TC), a plant native to Taiwan that belongs to the Scrophulariaceae family, has been used in traditional Chinese medicine to treat human diseases, including , stomatitis, hepatitis, pneumonia, and gastroenteritis, etc., owing to its detoxification, anti-inflammatory, and diuretic effects [21,22]. Experimentally, TC extracts have been reported to have excellent anti-inflammatory effects on macrophages and inhibitory effects on lipid deregulation in adipocytes [21,22]. Moreover, the components of TC, such as botulin, betulinic acid, and oleanolic acid, are reported to exert anti-inflammatory, anti-cancer, or anti-hyperglycemic activities [23–25]. Although the protective effects of TC extracts on inflammatory diseases have been examined extensively in in vitro and in vivo models [21,22,24]; there is little information about the role of TC extracts in endothelial dysfunction and related cardiovascular diseases. Further investigations of the vascular protective effects of TCEE and its underlying molecular mechanisms in eNOS/NO signaling and EC function are warranted. Given the impact of TCEE on inflammatory and metabolic diseases, the present study aimed to characterize the effects of TCEE on ECs and the molecular mechanisms underlying these effects. First, we investigated effects of TCEE and the related molecular mechanisms underlying the activation of eNOS-NO signaling, and then, we ascertained whether TCEE-mediated enhancement of NO bioavailability contributes to its anti-inflammatory property against inflammatory responses. Our third aim was to explore the effects of TCEE on the inflammatory response and the progression of / atherosclerosis in apolipoprotein E-deficient (ApoE− −) mice. Our results demonstrated that TECC promotes NO production via the Akt/CaMKII/AMPK/eNOS signaling cascade, which inhibits the inflammatory responses, and ultimately retards the progression of atherosclerosis.

2. Results

2.1. TCEE Increases NO Production by Increasing the Phosphorylation of eNOS in ECs To our knowledge, there is no information about the working concentrations of TCEE in ECs. For this reason, we thus tested the effect of TCEE on NO production and physiological function in ECs at a variety of concentrations ranging from 0.125 to 8 µg/mL. To test whether TCEE can activate the eNOS/NO signaling pathway, ECs were treated with the indicated concentrations (0.125, 0.25, 0.5, 1, 2, 4, and 8 µg/mL) of TCEE, and the effects of TCAE on cell viability and NO production were Int.Int. J.J. Mol.Mol. Sci. 20202020,, 2121,, 1532x FOR PEER REVIEW 33 ofof 1717

1, 2, 4, and 8 μg/mL) of TCEE, and the effects of TCAE on cell viability and NO production were examined. Treating the ECs with 0.125 to 8 µg/mL of TCEE for 24 h did not affect the cell viability examined. Treating the ECs with 0.125 to 8 μg/mL of TCEE for 24 h did not affect the cell viability (Figure1A–D) and increased the NO bioavailability (Figure1E). However, the eNOS protein level was (Figure 1A–D) and increased the NO bioavailability (Figure 1E). However, the eNOS protein level not affected in ECs following the treatment with 0.125 to 8 µg/mL TCEE for 24 h (Figure1F), suggesting was not affected in ECs following the treatment with 0.125 to 8 μg/mL TCEE for 24 h (Figure 1F), the involvement of increased eNOS activity in the TCEE-induced NO production. Indeed, the increase suggesting the involvement of increased eNOS activity in the TCEE-induced NO production. Indeed, in NO production was abolished by the pretreatment with L-NAME, a non-selective inhibitor of NOS the increase in NO production was abolished by the pretreatment with L-NAME, a non-selective (Figure1G), which indicates that the increase in NO bioavailability by TCEE was specifically linked to inhibitor of NOS (Figure 1G), which indicates that the increase in NO bioavailability by TCEE was the function of eNOS. Among the concentrations of TCEE, 2, 4, and 8 µg/mL of TCEE had the most specifically linked to the function of eNOS. Among the concentrations of TCEE, 2, 4, and 8 μg/mL of potent effect on NO production; however, there was no statistical significance between these three TCEE had the most potent effect on NO production; however, there was no statistical significance groups. We thus decided to use 2 µg/mL of TCEE for further experiments. Next, we examined the between these three groups. We thus decided to use 2 μg/mL of TCEE for further experiments. Next, effect of TCEE on eNOS phosphorylation at various time points. We found that TCEE induced the we examined the effect of TCEE on eNOS phosphorylation at various time points. We found that phosphorylation of eNOS at Ser635 and Ser1179 in a time-dependent manner without affecting its TCEE induced the phosphorylation of eNOS at Ser635 and Ser1179 in a time-dependent manner phosphorylation at Ser617 and Thr497 (Figure2A–D). These results suggest that TCEE increases the without affecting its phosphorylation at Ser617 and Thr497 (Figure 2A–D). These results suggest that NO bioavailability by increasing eNOS phosphorylation. TCEE increases the NO bioavailability by increasing eNOS phosphorylation.

Figure 1. Effects of Torenia concolor Lindley var. Formosama Yamazaki ethanol extracts (TCEE) on Figure 1. Effects of Torenia concolor Lindley var. Formosama Yamazaki ethanol extracts (TCEE) on cell cell viability and NO bioavailability in ECs. ECs were treated with the indicated concentrations viability and NO bioavailability in ECs. ECs were treated with the indicated concentrations (0.125-8 (0.125-8 µg/mL) of TCEE for 24 h. (A–C) The cell viability by MTT assay, CCK-8 assay, and LDH assay. μg/mL) of TCEE for 24 h. (A–C) The cell viability by MTT assay, CCK-8 assay, and LDH assay. (D) (D) Microscopic analysis of cells. (E) The level of in culture medium by Griess assay. (F) Western Microscopic analysis of cells. (E) The level of nitrite in culture medium by Griess assay. (F) Western blot analysis of total eNOS and α-tubulin. (G) ECs were pretreated with L-NAME (400 µM) for 2 h, blot analysis of total eNOS and α-tubulin. (G) ECs were pretreated with L-NAME (400 μM) for 2 h, and then with TCEE (2 µM) for 24 h. The level of nitrite in the culture medium was measured. Data and then with TCEE (2 μM) for 24 h. The level of nitrite in the culture medium was measured. Data are mean SEM from 5 independent experiments. * p < 0.05 vs. vehicle-treated group; # p < 0.05 vs. are mean ±± SEM from 5 independent experiments. * p < 0.05 vs. vehicle-treated group; # p < 0.05 vs. TCEE-treated group. TCEE-treated group.

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Figure 2. TCEE induces endothelial NO synthase (eNOS) activation in ECs. ECs were treated with TCEE Figure 2. TCEE induces endothelial NO synthase (eNOS) activation in ECs. ECs were treated with (2 µg/mL) for the indicated times. (A–D) Cells were lysed and subjected to Western blot analysis for TCEE (2 μg/mL) for the indicated times. (A–D) Cells were lysed and subjected to Western blot analysis quantifying the levels of phosphorylated eNOS (at Thr495, Ser617, Ser635, and Ser1177) or total eNOS for quantifying the levels of phosphorylated eNOS (at Thr495, Ser617, Ser635, and Ser1177) or total protein. Data are mean SEM from 5 independent experiments. * p < 0.05 vs. vehicle-treated group. eNOS protein. Data are± mean ± SEM from 5 independent experiments. * p < 0.05 vs. vehicle-treated 2.2. Activationgroup. of the Akt-CaMKII-AMPK Signaling Pathway Is Essential for the TCEE-Induced Phosphorylation of eNOS and NO Production 2.2. Activation of the Akt-CaMKII-AMPK Signaling Pathway Is Essential for the TCEE-Induced The Akt-CaMKII-AMPK signaling pathway is reported to play an important role in eNOS activation Phosphorylation of eNOS and NO Production and NO production [12]. Next, we examined whether the Akt-CaMKII-AMPK signaling pathway is involvedThe Akt in- TCEE-inducedCaMKII-AMPK eNOS signaling activation pathway and is NO reported production. to play Our an results important showed role that in TCEE eNOS time-dependentlyactivation and NO increased production the level[12]. ofNext, phosphorylation we examined of whether Akt, CaMKII, the Akt and-CaMKII AMPK,-AMPK which occurredsignaling atpathway 15 to 30 is min involved after in the TCEE treatment-induced (Figure eNOS3A–C). activation Inhibition and NO of production. the Akt-CaMKII-AMPK Our results showed signaling that pathwayTCEE time by- LY294002dependen (antly increased Akt inhibitor), the level KN62 of (a phosphorylation CaMKII inhibitor), of Akt, or compound CaMKII, and C (C.C., AMPK, an AMPK which inhibitor)occurred abrogatedat 15 to 30 the min TCEE-induced after the treatment NO production (Figure 3A (Figure–C). 3InhibitionD). By using of the these Akt pharmacological-CaMKII-AMPK inhibitors,signaling pathway the up- and by LY294002 downstream (an relationshipsAkt inhibitor), of KN62 the Akt, (a CaMKII CaMKII, inhibitor), and AMPK or compoun followedd by C TCEE (C.C., treatmentan AMPK were inhibitor) established abrogated (Figure the4A–C). TCEE Hence,-induced the activation NO production of the Akt-CaMKII-AMPK (Figure 3D). By using signaling these cascadepharmacological is required inhibitors, for TCEE-induced the up- and eNOS downstream activation relationships and NO production. of the Akt, CaMKII, and AMPK followed by TCEE treatment were established (Figure 4A–C). Hence, the activation of the Akt- CaMKII-AMPK signaling cascade is required for TCEE-induced eNOS activation and NO production.

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Figure 3. Akt-CaMKII-AMPK signaling is involved in the TCEE-mediated enhancement of NO Figure 3. Akt-CaMKII-AMPK signaling is involved in the TCEE-mediated enhancement of NO bioavailability in ECs. (A–C) ECs were treated with TCEE (2 µg/mL) for the indicated times. Western bioavailability in ECs. (A–C) ECs were treated with TCEE (2 μg/mL) for the indicated times. Western blot analysis of phosphorylated or total of Akt, CaMKII, and AMPK and α-tubulin. (D) ECs were blot analysis of phosphorylated or total of Akt, CaMKII, and AMPK and α-tubulin. (D) ECs were pretreated with LY294002 (10 µM), compound C (C.C., 10 µM), or KN62 (10 µM) for 2 h, and then with

TCEEpretreated (2 µg /mL)with forLY294002 24 h. Griess (10 μM), assay compound of the nitrite C level(C.C., in 10 theμM), culture or KN62 medium. (10 DataμM) for are 2 mean h, andSEM then with TCEE (2 μg/mL) for 24 h. Griess assay of the nitrite level in the# culture medium. Data are± mean Int. J. Mol. fromSci. 20 520 independent, 21, x FOR PEER experiments. REVIEW * p < 0.05 vs. vehicle-treated group; p < 0.05 vs. TCEE-treated group. 6 of 17 ± SEM from 5 independent experiments. * p < 0.05 vs. vehicle-treated group; # p < 0.05 vs. TCEE-treated group.

Figure 4. Cont.

Figure 4. The up- and downstream relationships of the Akt, CaMKII, and AMPK signaling cascade in TCEE-treated ECs. (A–C) ECs were pretreated with LY294002 (10 μM), compound C (C.C., 10 μM), or KN62 (10 μM) for 2 h, and then with TCEE (2 μg/mL) for 30 min. Western blot analysis of phosphorylated or total of Akt, CaMKII, AMPK, eNOS, and α-tubulin. Data are mean ± SEM from 5 independent experiments. * p < 0.05 vs. vehicle-treated group; # p < 0.05 vs. TCEE-treated group.

2.3. The Akt-CaMKII-AMPK Signaling Plays a Key Role in the TCEE-Mediated Promotion of EC Proliferation, Migration, and Tube Formation Under physiological conditions, increased NO levels can promote EC proliferation, migration, and tube formation, and lead to angiogenesis [26]. We then investigated the effects of TCEE on EC proliferation, migration, and tube formation, and whether the Akt-CaMKII-AMPK/eNOS/NO signaling cascade is involved in the proangiogenic effects of TCEE. Our results revealed that treatment with TCEE enhanced EC proliferation, migration, and tube formation, which were abolished by the pretreatment of the ECs with LY294002, KN62, or C.C. (Figures 5 and 6). These findings suggest that the activation of the Akt-CaMKII-AMPK/eNOS/NO signaling pathway plays a crucial role in the TCEE-mediated benefits associated with the physiological function of ECs.

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Figure 4. The up- and downstream relationships of the Akt, CaMKII, and AMPK signaling cascade in Figure 4. The up- and downstream relationships of the Akt, CaMKII, and AMPK signaling cascade in TCEE-treated ECs. (A–C) ECs were pretreated with LY294002 (10 µM), compound C (C.C., 10 µM), TCEE-treated ECs. (A–C) ECs were pretreated with LY294002 (10 μM), compound C (C.C., 10 μM), or KN62 (10 µM) for 2 h, and then with TCEE (2 µg/mL) for 30 min. Western blot analysis of or KN62 (10 μM) for 2 h, and then with TCEE (2 μg/mL) for 30 min. Western blot analysis of phosphorylated or total of Akt, CaMKII, AMPK, eNOS, and α-tubulin. Data are mean SEM from 5 phosphorylated or total of Akt, CaMKII, AMPK, eNOS, and α-tubulin. Data are mean ±± SEM from 5 independent experiments. * p < 0.05 vs. vehicle-treated group; # p < 0.05 vs. TCEE-treated group. independent experiments. * p < 0.05 vs. vehicle-treated group; # p < 0.05 vs. TCEE-treated group. 2.3. The Akt-CaMKII-AMPK Signaling Plays a Key Role in the TCEE-Mediated Promotion of EC Proliferation, Migration,2.3. The Akt and-CaMKII Tube Formation-AMPK Signaling Plays a Key Role in the TCEE-Mediated Promotion of EC Proliferation, Migration, and Tube Formation Under physiological conditions, increased NO levels can promote EC proliferation, migration, and tubeUnder formation, physiological and leadconditions, to angiogenesis increased [ 26NO]. Welevels then can investigated promote EC the proliferation, effects of TCEE migration, on EC proliferation,and tube formation, migration, and and lead tube to formation,angiogenesis and [26]. whether We then the Akt-CaMKII-AMPKinvestigated the effects/eNOS of/ NOTCEE signaling on EC cascadeproliferation, is involved migration, in the proangiogenic and tube formation, effects of TCEE. and whether Our results the revealed Akt-CaMKII that treatment-AMPK/eNOS/NO with TCEE enhancedsignaling EC cascade proliferation, is involved migration, in the and proangiogenic tube formation, effects which of were TCEE. abolished Our results by the revealed pretreatment that oftreatment the ECs with with LY294002, TCEE enhanced KN62, or EC C.C. proliferation, (Figures5 and migration,6). These findings and tube suggest formation, that the which activation were ofabolished the Akt-CaMKII-AMPK by the pretreatment/eNOS of/ NOthe signalingECs with pathwayLY294002, plays KN62, a crucial or C.C. role (Figures in the TCEE-mediated5 and 6). These benefitsfindingsInt. J. Mol. associated suggestSci. 2020, 2that1, withx FOR the the PEERactivation physiological REVIEW of the functionAkt-CaMKII of ECs.-AMPK/eNOS/NO signaling pathway plays7 of 17a crucial role in the TCEE-mediated benefits associated with the physiological function of ECs.

Figure 5. Cont.

Figure 5. The signaling cascade involved in the TCEE-induced promotion of EC proliferation and migration. ECs were pretreated with LY294002 (10 μM), compound C (C.C., 10 μM), or KN62 (10 μM) for 2 h, then with TCEE (2 μg/mL) for 8 h. (A) MTT assay of EC proliferation. (B,C) EC migration was evaluated by the trans-well assay after TCEE treatment. The number of migrating cells was counted compared to the case for the vehicle-treated cells, and the images were generated by microscopy. Data are mean ± SEM from 5 independent experiments. * p < 0.05 vs. vehicle-treated group; # p < 0.05 vs. TCEE-treated group.

Figure 6. The signaling cascade involved in TCEE-induced promotion of EC tube formation. ECs were pretreated with LY294002 (10 μM), compound C (C.C., 10 μM), or KN62 (10 μM) for 2 h or L-NAME (400 μM) for 1 h, then with TCEE (2 μg/mL) for 8 h. (A,B) ECs were cultured in the attachment matrix in the indicated treatments. Tube formation was visualized; the bar graphs indicate the branch points as a fold of those in case of the vehicle-treated control in five randomly selected microscopic fields.

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FigureFigure 5. 5.The The signalingsignaling cascadecascade involvedinvolved inin the the TCEE-induced TCEE-induced promotion promotion of of EC EC proliferation proliferation and and Figure 5. The signaling cascade involved in the TCEE-induced promotion of EC proliferation and migration.migration. ECs ECs were were pretreated pretreated with with LY294002 LY294002 (10 (10µ μM),M), compound compound C C (C.C., (C.C., 10 10µ μM),M), or or KN62 KN62 (10 (10µ μM)M) migration. ECs were pretreated with LY294002 (10 μM), compound C (C.C., 10 μM), or KN62 (10 μM) forfor 2 2 h, h, then then with with TCEE TCEE (2 (2µ μg/mL)g/mL) for for 8 8 h. h. ( A(A)) MTT MTT assay assay of of EC EC proliferation. proliferation. ( B(B,C,C)) EC EC migration migration was was for 2 h, then with TCEE (2 μg/mL) for 8 h. (A) MTT assay of EC proliferation. (B,C) EC migration was evaluatedevaluated by by the the trans-well trans-well assay assay after after TCEE TCEE treatment. treatment. The The number number of of migrating migrating cells cells was was counted counted evaluated by the trans-well assay after TCEE treatment. The number of migrating cells was counted comparedcompared to to the the case case for for the the vehicle-treated vehicle-treated cells, cells, and and the the images images were were generated generated by by microscopy. microscopy. Data Data # arecomparedare mean mean to±SEM SEMthe case from from for 5 5 independentthe independent vehicle-treated experiments. experiments. cells, and * * pthe p< < images0.05 0.05 vs. vs. were vehicle-treated vehicle generated-treated by group; group; microscopy.# p p< <0.05 0.05 Data vs.vs. ± # TCEE-treatedareTCEE mean-treated ± SEM group. group. from 5 independent experiments. * p < 0.05 vs. vehicle-treated group; p < 0.05 vs. TCEE-treated group.

Figure 6. The signaling cascade involved in TCEE-induced promotion of EC tube formation. ECs were pretreatedFigure 6. The with signaling LY294002 cascade (10 µM), involved compound in TCEE C (C.C.,-induced 10 µ promotionM), or KN62 of EC (10 tubeµM) formation for 2 h or .l ECs-NAME were (400FigurepretreatedµM) 6. forThe with 1 signaling h, thenLY294002 with cascade TCEE(10 μM),involved (2 µ compoundg/mL) in TCEE for 8 C h.-induced (C.C., (A,B) 10 ECs promotion μM), were or culturedKN62 of EC (10 tube in μM) the formation attachmentfor 2 h or. ECs L- matrixNAME were inpretreated(400 the μM) indicated forwith 1 h, treatments.LY294002 then with (10 TubeTCE μM),E formation (2 compound μg/mL) was for C visualized;8 (C.C., h. (A, B10) ECsμM), the were baror KN62 graphs cultured (10 indicate μM)in the for theattachment 2 branchh or L-NAME points matrix as(400in a the foldμM) indicated offor those 1 h, treatments.then in case with of TCE the TubeE vehicle-treated (2 formation μg/mL) for was 8 control h.visualized; (A,B in) ECs five the were randomly bar cultured graphs selected indicatein the microscopicattachment the branch matrix fields.points Datainas the a arefold indicated mean of those treatments.SEM in case from of 5Tube independentthe vehicleformation-treated experiments. was visualized;control *inp

2.4. Activation of eNOS Is Essential for the Anti-Inflammatory Effects of TCEE TCEE has been reported to have excellent anti-inflammatory effects [21,22]. Based on the above findings, we thus investigated whether eNOS activation mediates the TCEE-conferred anti-inflammatory effects. We demonstrated that tumor necrosis factor-α (TNF-α) increased the expression of ICAM-1 and VCAM-1, as well as monocyte adhesion onto ECs (Figure7A–E). However, these pro-inflammatory responses elicited by TNF-α were abrogated by the NOS inhibitor L-NAME Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 8 of 17

Data are mean ± SEM from 5 independent experiments. * p < 0.05 vs. vehicle-treated group; # p < 0.05 vs. TCEE-treated group.

2.4. Activation of eNOS Is Essential for the Anti-Inflammatory Effects of TCEE TCEE has been reported to have excellent anti-inflammatory effects [21,22]. Based on the above findings, we thus investigated whether eNOS activation mediates the TCEE-conferred anti- Int.inflammatory J. Mol. Sci. 2020 , effects.21, 1532 We demonstrated that tumor necrosis factor-α (TNF-α increased8 of the 17 expression of ICAM-1 and VCAM-1, as well as monocyte adhesion onto ECs (Figure 7A–E). However, (Figurethese pro7A–E).-inflammatory These findings responses suggest elicited the by importance TNF-α were of eNOSabrogated activation by the NOS for TCEE inhibitor to elicit L-NAME its anti-inflammatory(Figure 7A–E). These action findings in ECs. suggest the importance of eNOS activation for TCEE to elicit its anti- inflammatory action in ECs.

Figure 7. TCEE inhibits the TNFα-induced adhesion of monocytes onto ECs and the protein expression Figure 7. TCEE inhibits the TNFα-induced adhesion of monocytes onto ECs and the protein of adhesion molecules in ECs. ECs were pretreated with TNFα (10 ng/mL) or the vehicle for 12 h, expression of adhesion molecules in ECs. ECs were pretreated with TNFα (10 ng/mL) or the vehicle followed by treatment with L-NAME (400 µM) for 1 h and/or TCEE (2 µg/mL) for another 18 h. (A–C)

Cellularfor 12 h, lysates followed were by subjectedtreatment towith Western L-NAME blot (400 analysisμM) for 1 quantifying h and/or TCEE the protein(2 μg/mL) expression for another of ICAM-1,18 h. (A VCAM-1,–C) Cellular and lysatesα-tubulin. were (D subjected,E) BCECF-AM-labeled to Western blot human analysis monocyte for quantifying THP-1 cells the (1 protein105) × wereexpress addedion andof ICAM incubation-1, VCAM with-1, ECs and for α- 1tubulin. h. The ( cellularD,E) BCECF lysates-AM were-labeled subjected human to fluorometrymonocyte THP and-1 5 photo-micrographed.cells (1 × 10 ) were added Data areand mean incubationSEM with from ECs 5 independent for 1 h. The experiments. cellular lysates * p

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Figure 8. TCEE ameliorates hyperlipidemia and atherosclerosis in ApoE−/− mice. Male ApoE−/− mice at / / ageFigureFigure 16 8. weeks 8. TCEETCEE were ameliorates orally administered hyperlipidemia hyperlipidemia with and and TCEE atherosclerosis atherosclerosis (5 mg/kg/day) in inApoE ApoE or−/− vehicle −mice− mice.. Male for Male ApoE 4 weeks. ApoE−/− mice (−A− )at Atheroscleroticmiceage at 16 age weeks 16 weekslesions were wereorallyat aortic orally administered roots administered were stained with with TCEE with TCEE (5H&E. mg/kg/day) (5 ( mgB) The/kg/ day)area or vehicle orof vehiclemacrophages for for4 weeks. 4 weeks.in the (A ) - atherosclerotic(AAtherosclerotic) Atherosclerotic lesions. lesions lesions (atC ataorticG aortic) Serum roots roots were levels were stained of stained total with with cholesterol, H&E. H&E. (B) ( BThe non) The area-high area of-density ofmacrophages macrophages lipoprotein in inthe cholesteroltheatherosclerotic atherosclerotic (non- HDL lesions. lesions.-c), HDL(C (-CG cholesterol–)G ) Serum Serum levels(HDL levels -c), of of and total total triglyc cholesterol, cholesterol,erides. Data non-high-density non are-high mean-density ± SEM lipoprotein lipoprotein from 10 mice.cholesterol * p < 0.05 (non-HDL-c), vs. vehicle. HDLScale cholesterolbar = 300 μm. (HDL-c), and triglycerides. Data are mean SEM from cholesterol (non-HDL-c), HDL cholesterol (HDL-c), and triglycerides. Data are mean ±± SEM from 10 µ 10mice. mice. * *p p< <0.050.05 vs. vs. vehicle. vehicle. Scale Scale bar bar = 300= 300 μm.m.

/ / Figure 9. TCEE attenuates systemic and aortic inflammation in ApoE− − mice. Male ApoE− − mice at age 16 weeks were orally administered with TCEE (5 mg/kg/day) or vehicle (oil) for 4 weeks. (A–E) Figure 9. TCEE attenuates systemic and aortic inflammation in ApoE−/− mice. Male ApoE−/− mice at ELISA of serum levels of TNF-α, IL-1β, MCP-1, MIP-2 and IL-6. (F) Western blot analysis of protein age 16 weeks were orally administered with TCEE (5 mg/kg/day)/ or vehicle−/− (oil) for 4 weeks.−/− (A-E) levelsFigure of ICAM-1,9. TCEE attenuates VCAM-1, andsystemic -α tubulin and aortic in aortas inflammation from ApoE −in− ApoEmice. Datamice. are Male mean ApoESEM mice from at ELISA of serum levels of TNF-α, IL-1β, MCP-1, MIP-2 and IL-6. (F) Western blot analysis± of protein 10age mice. 16 weeks * p < 0.05 were vs. orally vehicle. administered with TCEE (5 mg/kg/day) or vehicle (oil) for 4 weeks. (A-E) −/− levelsELISA of ofICAM serum-1, levelsVCAM of-1, TNF and- α,-α IL tubulin-1β, MCP in aortas-1, MIP from-2 and ApoE IL-6. (mice.F) Western Data areblot mean analysis ± SEM of protein from 3. Discussion 10levels mice. of * pICAM < 0.05- 1,vs. VCAM vehicle.-1, and -α tubulin in aortas from ApoE−/− mice. Data are mean ± SEM from TCEE10 mice. is known * p < 0.05 to havevs. vehicle. anti-inflammatory, anti-allergic, and lipid-lowering effects [21,22]; however, 3. Discussion its biological impact on cardiovascular diseases remains unclear. In this study, we demonstrated that TCEE3. DiscussionTCEE increased is known eNOS to phosphorylation, have anti-inflammatory, NO production, anti-allergic, and angiogenic and lipid responses-lowering in effe EA.hy926cts [21,22]; ECs however,throughTCEE the its Akt is biological known/CaMKII to/ impactAMPK have anti signalingon - cardiovascularinflammatory, pathway. antiExposure diseases-allergic, remains of ECs and to lipid unclear. TCEE-lowering rapidly In this effeincreased study,cts [21,22]; thewe demonstratedphosphorylationhowever, its thatbiological of eNOS TCEE atimpact increased Ser635 on and cardiovascular eNOS Ser1179, phosphorylation, and further diseases increased remains NO production, NO unclear. production. In and this In angiogenic study, addition, we responsesthedemonstrated phosphorylation in EA.hy926 that of TCEE Akt,ECs through CaMKII, increased the and Akt/CaMKII/AMPK eNOS AMPK phosphorylation, is involved in signaling TCEE-induced NO p production,athway. NO Exposure production, and angiogenicof ECs which to TCEEisresponses consistent rapidly in with increasedEA.hy926 the results theECs phosphorylation ofthrough studies the by Akt/CaMKII/AMPK Ching of eNOS et al. at and Ser635 Guo signaling etand al., Ser1179, who pathway. found and thatExposurefurther Akt, increased CaMKII,of ECs to TCEE rapidly increased the phosphorylation of eNOS at Ser635 and Ser1179, and further increased

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 10 of 17 Int. J. Mol. Sci. 2020, 21, 1532 10 of 17 NO production. In addition, the phosphorylation of Akt, CaMKII, and AMPK is involved in TCEE- induced NO production, which is consistent with the results of studies by Ching et al. and Guo et al., and AMPK are the key kinases in eNOS phosphorylation [26,27]. NO is known to be the most who found that Akt, CaMKII, and AMPK are the key kinases in eNOS phosphorylation [26,27]. NO important modulator in regulating the physiological functions of ECs, such as proliferation, migration, is known to be the most important modulator in regulating the physiological functions of ECs, such and angiogenesis [28,29]. Indeed, our findings showed that TCEE promoted EC proliferation, migration, as proliferation, migration, and angiogenesis [28,29]. Indeed, our findings showed that TCEE and angiogenesis in an NO-dependent manner. Moreover, NO has an anti-inflammatory action and promoted EC proliferation, migration, and angiogenesis in an NO-dependent manner. Moreover, NO thus, inhibits the activation of circulating leukocytes [30]. Moreover, TCEE inhibited the TNFα-induced has an anti-inflammatory action and thus, inhibits the activation of circulating leukocytes [30]. upregulation of the adhesion molecules ICAM-1 and VCAM-1, as well as the monocyte adhesion onto Moreover, TCEE inhibited the TNFα-induced upregulation of the adhesion molecules ICAM-1 and ECs. Notably, these beneficial effects of TCEE can also be observed in primary human microvascular VCAM-1, as well as the monocyte adhesion onto ECs. Notably, these beneficial effects of TCEE can ECs (Figures S1 and S2). Our in vivo findings further support this notion by evidencing that TCEE also be observed in primary human microvascular ECs (Figures S1 and S2). Our in vivo findings decreased the systemic and aortic inflammation and thus delayed the progression of atherosclerosis further support/ this notion by evidencing that TCEE decreased the systemic and aortic inflammation in ApoE− − mice. Collectively, TCEE may exert its anti-inflammatory and anti-atherogenic effects by and thus delayed the progression of atherosclerosis in ApoE−/− mice. Collectively, TCEE may exert its preventing EC dysfunction (Figure 10). anti-inflammatory and anti-atherogenic effects by preventing EC dysfunction (Figure 10).

Figure 10. Schematic illustration of the proposed mechanism underlying the TCEE-mediated Figureanti-inflammatory 10. Schematic eff ectillustration in ECs andof the atherosclerosis. proposed mechanism As shown, underlying challenge the withTCEE TCEE-mediated elicits anti the- inflammatoryactivation ofthe effect Akt in/CaMKII ECs and/AMPK athero/sclerosis.eNOS/NO As pathway, shown, challenge which in with turn TCEE promotes elicits pro-angiogenic the activation ofactivity the Akt/CaMKII/AMPK/eNOS/NO and inhibits the pro-inflammatory pathway, response which by TNF- in turnα in promotes ECs and retardspro-angiogenic the atherosclerosis activity and in / −/− ApoEinhibits− − themice. pro-inflammatory response by TNF-α in ECs and retards the atherosclerosis in ApoE mice. Notably, EC dysfunction is the key event in the early stage of cardiovascular diseases, including hypertensionNotably, EC and dysfunction atherosclerosis is the [31 key,32 event]. It isin characterized the early stage by of decreasedcardiovascular NO bioavailabilitydiseases, including and hypertensionincreased expression and atherosclerosis of adhesion [31,32]. molecules It is in characterized atherosclerosis by [33 decreased,34]. Modulating NO bioavailability the activation and increasedof eNOS-NO expression signaling of hasadhesion been suggestedmolecules asin aatheros therapeuticclerosis strategy [33,34].for Modulating treating or the preventing activation the of progressioneNOS-NO signaling of cardiovascular has been diseases suggested [4, 35 as]. a For therapeutic instance, strategy Xing et al. for and treating Li. et or al. preventing have reported the progressionthat supplementation of cardiovascular with resveratrol diseases or[4,35]. salidroside For instance, improves Xing endothelial et al. and Li. dysfunction et al. have andreported alleviates that / supplemeatherosclerosisntation in ApoE with − resveratrol− mice [36 ,37 or]. salidroside Furthermore, improves treatment endothelial with Morus dysfunction alba extract activates and alleviates eNOS atherosclerosissignaling and maintains in ApoE blood−/− mice pressure [36,37]. homeostasis Furthermore, in mice treatment [38]. Our with previous Morus study alba hasextract demonstrated activates eNOSthat capsaicin signaling and and evodiamin, maintains which blood are thepressure agonists homeostasis of transient in receptor mice [38]. potential Our vanilloid previous 1, study retard has the progressiondemonstrated of atherosclerosisthat capsaicin and by activating evodiamin, the which eNOS/ NOare signalingthe agonists pathway of transient [13,15, 26receptor,39]. In potential addition, vanilloidstatins, which 1, retard are lipid-loweringthe progression clinical of atherosclerosis drugs, confer by cardiovascular activating the benefitseNOS/NO via signaling pleiotropic pathway effects, [13,15,26,39].such as increased In addition, NO production , which and anti-inflammatory are lipid-lowering activities clinical [8 drugs,,40]. In confer this study, cardiovascular our data benefitsconfirmed via this pleiotropic notion byeffects, evidencing such as that increased TCEE activatesNO production the eNOS-NO and anti- signalinginflammatory pathway activities and [8,40].attenuates In this inflammatory study, our data responses; confirmed however, this notion the eff ectsby evidencing of TCEE on that atherosclerosis TCEE activates and the hypertension, eNOS-NO signaling pathway and attenuates inflammatory responses; however, the effects of TCEE on

Int. J. Mol. Sci. 2020, 21, 1532 11 of 17 as well as the molecular mechanisms underlying these effects, are still unknown. To this end, investigations targeting the key events in the development of atherosclerosis are warranted. Importantly, previous studies have reported that 11 bioactive components, including lupeol, stigmasterol, β-sitosterol, betulin, betulinic acid, oleanolic acid, maslinic acid, alphitolic acid, 3-epimaslinic acid, augustic acid, and β-sitosterol-3-0-β-d-glucoside, are found in TC extracts [21,24,25]. Moreover, growing evidence has suggested that these components have protective effects against cardiovascular and inflammatory diseases [11,41]. For example, lupeol is reported to have cardioprotective and anti-inflammatory effects by downregulating the expression of TNF-α, IL-2, IL-4, IL-5, IL-6, IL-11, and prostaglandin E2 [11,41]. Impaired of phytosterols, including β-sitosterol and stigmasterol, causes phytosterolemia, which is closely related to early atherosclerosis [10,42]. Supplementation with phytosterols retards the progression of atherosclerosis by reducing the cholesterol absorption [10,42]. Although these studies have pointed out the beneficial effects of many components of TC extracts, in this study, we cannot ascertain which component accounts for the TCEE-mediated enhancement of NO bioavailability and anti-inflammatory effects in ECs. Therefore, further investigation is needed for studying the protective effects of TCEE components and their regulatory mechanisms in cardiovascular diseases. Moreover, the purification methods by which the TC extracts are isolated may affect their bioactivity [21–25]. Previous studies have reported that TC extracts can be purified using water, ethanol, acetate extract, and n-butanol [23–25]. These TC extracts may have a variety of effects in regulating the physiological functions of the cardiovascular system and the development of cardiovascular diseases [22,24]. For instance, all extracts obtained by various purification methods have been shown to have a similar inhibitory effect on lipopolysaccharide-induced NO production in macrophages [22,24]; however, compared with other TC extracts, the TC extract purified using n-butanol has the strongest efficacy in inhibiting lipid accumulation via the activation of peroxisome proliferator-activated receptorγ in adipocytes [22]. However, the effects of other TC extracts, including TCEAE, TCBUE, and TCWE, on the NO-mediated regulation of EC functions and the underlying mechanism require further investigations. Because the hydrophobic property of TCEE facilitates its absorption into cells, compared to the case for hydrophilic extracts, we used TCEE throughout our study. Endothelium-derived NO plays an important role in regulating the vascular homeostasis, and has a protective role against the deregulation of lipid metabolism and inflammation, which are the hallmarks of cardiovascular and metabolic diseases [43–45]. Our findings further confirmed the beneficial effects of TCEE on vascular biology via the enhancement of NO bioavailability. Collectively, these observations from our study or others suggest that TC extracts may have therapeutic potential for treating inflammatory and metabolic diseases. However, our study contains the limitation that we only used the antibodies to detect the phosphorylation sites of eNOS; however, the antibodies may have nonspecific cross-reaction on various target sites. The kinase assay using 32P labelling of eNOS could be the more precise method and further support our findings regarding the status of eNOS phosphorylation. Nevertheless, we did not detect eNOS phosphorylation by the kinase assay due to the reason that our labs are not certified to perform experiments using radioactive substance. On the other hand, we did not study the mechanism how TCEE starts the signaling cascade in this study. Whether there is a specific receptor or target protein to TCEE requires further investigations. In conclusion, this study demonstrates that TCEE has the beneficial effects on angiogenesis and the inhibition of inflammation, by activating the eNOS-NO signaling pathway, which confers protective effects from atherosclerosis. Our study provides advanced information about the beneficial effects of TCEE in regulating endothelial function, inflammatory responses, and the progression of atherosclerosis. These findings may broaden the biological significance and biomedical implications of TCEE in the treatment of cardiovascular and metabolic diseases. Int. J. Mol. Sci. 2020, 21, 1532 12 of 17

4. Materials and Methods

4.1. Reagents Mouse antibody for α-tubulin, LY294002, Griess reagent, 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay kit, N-nitro-L- methyl ester (L-NAME), and phosphatase inhibitor cocktails 1 and 2 were obtained from Sigma-Aldrich (St. Louis, MO, USA). Compound C was procured from Calbiochem (San Diego, CA, USA). Rabbit antibodies for phosphor-eNOS at Ser617, 635, and 1179, phosphor-eNOS at Thr497, phosphor-Akt at Ser473, phosphor-CaMKII at Thr286, phosphor-AMPK at Thr172, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) were purchased from Technology (Beverly, MA, USA). Rabbit antibodies for eNOS, Akt, CaMKII, and AMPK were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Matrigel was procured from BD Biosciences (San Jose, CA, USA). ELISA kits for cytokines were obtained from R&D systems (Minneapolis, MN, USA). Cholesterol and triglyceride assay kits were from Randox (Crumlin, County Antrim, UK).

4.2. Cell Culture EA.hy926 ECs and THP-1 cells were obtained from the ATCC (Manassas, VA, USA) and cultured in DMEM or RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin (HyClone, Logan, UT, USA). EA.hy926 cells were grown until 100% confluence and then used for further experiments. Human microvascular ECs (HMECs) were obtained from the Centers for Disease Control (Atlanta, GA, USA) and grown in Medium 200 (Cascade Biologics Inc., Portland, OR, USA) supplemented with low serum growth supplement containing 2% FBS, 1 µg/mL hydrocortisone, 10 ng/mL human epidermal growth factor, 3 ng/mL human fibroblast growth factor, and 10 µg/mL heparin.

4.3. TC Extraction The TC was crushed to a powder and then was extracted with 95% ethanol followed by partition extraction by different solvents. For the ethanol extraction, TC powder was first soaked in 95% ethanol for 3 days and then the ethanol was removed by a vacuum pump to produce analytes at concentrate form. The concentrated crude extract was weighed and degassed by the nitrogen gas bubbling to remove the residual ethanol. For partition extraction, the aliquot of the crude extract was re-dissolved with ddH2O and placed into a partition-extraction bottle, and ethyl acetate and n-butanol were sequentially added, in the order of increasing solvent polarity. The two resulting organic extracts were referred to as TCEAE, composed of TC and ethyl acetate, and TCBUE, composed of TC and n-butanol. The concentrated aqueous phase remaining after the extraction was referred to as TCWE, which was composed of TC and water. The ethanol-free, concentrated extract was not used in the partition extraction, which was referred to as TCEE. The organic extracts TCEAE, TCBUE, and TCEE were re-dissolved in absolute ethanol and the TCWE extract was mixed with PBS to generate the stock solutions. All the stock solutions were filtered to remove endotoxin by ELGA LabWater Biofilter and stored at 20 C until further use. In this study, we only examined the effect of TCEE on the − ◦ physiological functions of ECs.

4.4. Cell Viability Assay MTT assay, cell counting kit-8 (CCK-8) assay, and lactate dehydrogenase (LDH) assay were performed according to the manufacturer’s instructions for evaluating the cell viability. First, the cells were incubated with or without indicated concentrations of TCEE for 24 h; MTT reagent was then added to the cells, followed by incubation for 3 h. On the other hand, the cultured medium was mixed with corresponding reagents of CCK-8 assay and LDH assay for 30 min. The absorbance of these samples was measured at 570 (MTT assay) and 490 nm (CCK-8 and LDH assays), respectively. The cell Int. J. Mol. Sci. 2020, 21, 1532 13 of 17 viability was normalized relative to the cellular protein concentration. Cells incubated with the vehicle were considered 100% viable.

4.5. Protein Extraction and Western Blot Analysis Cells were lysed with the SDS lysis buffer, which contained 1% Triton, 0.1% SDS, 0.2% sodium azide, 0.5% sodium deoxycholate, and proteinase inhibitors (1 mmol/L PMSF, 10 mg/mL aprotinin, and 1 mg/mL leupeptin). The lysates were centrifuged at 12,000 rpm for 5 min and the resulting supernatants were collected. The extracted protein was quantified by protein assay. The proteins were separated by SDS-PAGE; the protein bands were transferred onto BioTrace PVDF membranes. After being blocked with 5% skim milk, the blots were incubated with the primary antibodies, and then with the secondary antibodies. The protein bands were detected by using an enhanced chemiluminescence kit and the level of protein expression was quantified by using ImageQuant 5.2 (Healthcare biosciences, Philadelphia, PA, USA).

4.6. Determination of Nitrite Production Nitric oxide (NO) has an extremely short half-life and is quickly metabolized into nitrite and . Thus, the level of nitrite was determined by the Griess assay to assess NO production. The cell culture medium was mixed with an equal volume of Griess reagent, and then azo dye production was determined by measuring the absorbance of these samples at 540 nm after 15 min of incubation at room temperature. The level of nitrite was normalized relative to the cellular protein concentration. Sodium nitrite was used as a standard.

4.7. Cell Proliferation Assay EA.hy926 cells were cultured on 12-well plates in DMEM containing 10% FBS and subjected to serum starvation for 12 h. After treatment, the cells were labeled BrdU with for 4 h. BrdU incorporation was measured at least in triplicate at each time by the use of a cell proliferation ELISA colorimetric kit (Roche, Mannheium, Germany). The absorbance was measured by spectrophotometry at 370 nm and referred at 492 nm.

4.8. Cell Migration Assay The transwell migration assay was performed using modified chambers inserted into 24-well plates. First, 5 106 cells suspended in 300 µL of DMEM were added into the upper chamber. On the × other hand, 500 µL of DMEM was added into the lower chamber. After 18 h of incubation, the medium and unmigrated cells in the upper chamber were removed, and the migrated cells in the lower side of the membranes were stained with crystal violet. Images were digitally captured under a Nikon TE2000-U florescence microscope with an image analysis system (QCapture Pro 6.0, QImaging, Surrey, BC, Canada).

4.9. Matrigel Angiogenesis Assay

Matrigel was coated onto 3.5 cm dishes and allowed to polymerize for 30 min at 37 ◦C. ECs were seeded onto the Matrigel layer and subjected to the indicated treatments for 9 h. The tube formation ability of the ECs was quantified by counting the number of branch points and the images were photomicrographed under a Nikon TE2000-U florescence microscope with an image analysis system.

4.10. In Vitro Mononuclear-Endothelial Cell Adhesion Assay The adherence of THP-1 cells onto the ECs was examined under static conditions. For fluorescence staining, THP-1 cells were incubated with BCECF-AM at 37 ◦C for 1 h and then added into the culture medium at a concentration of 1 105 cells/mL; the PHP-1 cells were incubated with the ECs for × 1 h. Fluorescence of the cell lysates was measured by fluorometry (Molecular Devices, Sunnyvale, Int. J. Mol. Sci. 2020, 21, 1532 14 of 17

CA, USA) with excitation and emission wavelengths of 485 and 525 nm, respectively. Images were photomicrographed under a Nikon TE2000-U microscope with an image analysis system.

4.11. Serum Lipid Profile Analysis The serum levels of total cholesterol, high-density lipoprotein cholesterol (HDL-c), non-HDL-c, and triglycerides were measured by use of the blood biochemical analyzer Spotchem EZ SP 4430 (ARKRAY, Inc., Kyoto, Japan) or assay kits.

4.12. Measurement of Inflammatory Cytokines The serum concentrations of pro-inflammatory cytokines, including TNF-α, IL-1β, MCP-1, IL-6, and MIP-2, were measured by use of ELISA kits.

4.13. Mice All animal experiments were approved by the Animal Care and Utilization Committee of National Yang-Ming University and the investigation conformed to the Guide for the Care and Use of Laboratory / Animals (Institute of Laboratory Animal Resources, eighth edition, 2011). ApoE− − mice were purchased / from Jackson Laboratory (Bar Harbor, Maine). ApoE− − mice at 16 weeks old received oral daily treatment with TCEE (5 mg/kg body weight) or vehicle by gastric gavage for 4 weeks. At the end of the experiment, mice were euthanized with CO2 and the hearts were isolated and fixed with 4% paraformaldehyde and then embedded in paraffin. The deparaffinized sections were subjected to H&E staining and the quantification of atherosclerotic lesions at the aortic sinus was performed. The lysates of atherosclerotic aortas were subjected to Western blot analysis to evaluate the expression of proteins.

4.14. Statistical Analysis Results are presented as the mean SEM from five independent experiments or 10 mice. ± Mann–Whitney test was used to compare two independent groups. Kruska–Wallis test, followed by the Bonferroni post hoc analyses, were used for comparing data from multiple groups. SPSS software v8.0 (SPSS Inc. Chicago, IL, USA) was used to perform the statistical analyses. p < 0.05 was considered statistically significant.

Supplementary Materials: The following are available online at http://www.mdpi.com/1422-0067/21/4/1532/s1. Author Contributions: Conceptualization, T.-S.Y. and T.-S.L.; methodology, L.-C.C., B.-C.G. and C.-H.C.; formal analysis, L.-C.C., B.-C.G.; investigation, L.-C.C., B.-C.G., C.-H.C.; data curation, L.-C.C. and B.-C.G.; writing—original draft preparation, C.-J.C., T.-S.Y. and T.-S.L.; supervision, T.-S.L.; funding acquisition, L.-C.C. and T.-S.L. All authors have read and approved to the published version of the manuscript. Funding: This research was funded by the Ministry of Science and Technology, Taiwan and the Chang Gung Medical Research Foundation, Taiwan, grant number 105-2320-B-010-036, 105-2811-B-010-022, 106-2320-B-002-057-MY3, 106-2320-B-002-056 and 106-2811-B-002-146 were funded by the Ministry of Science and Technology, Taiwan and grant number CMRPF1C0211, CMRPF1C0212, and CMRPF1C0213 were funded by the Chang Gung Medical Research Foundation, Taiwan. Acknowledgments: The authors would like to thank Chwan-Fwu Lin for his assistance in the preparation of TCEE. Conflicts of Interest: The authors declare no conflict of interest.

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