# Curcumin modulates endothelial permeability and monocyte transendothelial migration by affecting endothelial cell dynamics

Laurent-Emmanuel Monfoulet, Sylvie Mercier, Dominique Bayle, Radu Tamaian, Nicolas Barber Chamoux, Christine Morand, Dragan Milenkovic

<figure><img src="image_1.png" /></figure>

## To cite this version:

Laurent-Emmanuel Monfoulet, Sylvie Mercier, Dominique Bayle, Radu Tamaian, Nicolas Barber Chamoux, et al.. Curcumin modulates endothelial permeability and monocyte transendothelial migration by affecting endothelial cell dynamics. Free Radical Biology and Medicine, 2017, 112, pp.109-120. 10.1016/j.freeradbiomed.2017.07.019. hal-01605170

HAL Id: hal-01605170

https://hal.science/hal-01605170

Submitted on 21 Feb 2023

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_2.png" /></figure>
1

Curcumin modulates endothelial permeability and monocyte

2

transendothelial migration by affecting endothelial cell dynamics

3

4

Laurent-Emmanuel Monfoulet<sup>a,∗</sup>, Sylvie Mercier<sup>a</sup>, Dominique Bayle<sup>a</sup>, Radu Tamaian<sup>b,c</sup>, Nicolas

5

Barber-Chamoux<sup>a,d</sup>, Christine Morand<sup>a</sup>, Dragan Milenkovic<sup>a</sup>

6

7

<sup>a</sup>Université Clermont Auvergne, INRA, UNH, CRNH Auvergne, F-63000 Clermont-Ferrand, France

8

<sup>b</sup> Research and Development Department, National Institute for Research and Development for

9

Cryogenic and Isotopic Technologies, RO-240050 Râmnicu Vâlcea, Romania

10

<sup>c</sup> SC Biotech Corp SRL, RO-240050 Râmnicu Vâlcea, Romania

11

<sup>d</sup> CHU Clermont-Ferrand, Service de Cardiologie, F-63000 Clermont-Ferrand, France.

12

13

**Corresponding Author**

14

<sup>∗</sup>Dr Laurent-Emmanuel Monfoulet

15

INRA

16

Unité de Nutrition Humaine (UNH) – UMR1019

17

Centre Auvergne-Rhône-Alpes

18

Site de Theix

19

63122 Saint Genès Champanelle

20

Email: laurent-emmanuel.monfoulet@inra.fr

21

Phone: +33 4 73 62 42 90

22

Fax: +33 4 73 62 46 38
23 **ABSTRACT**

24 Curcumin is a phenolic compound that exhibits beneficial properties for cardiometabolic health. We 25 previously showed that curcumin reduced the infiltration of immune cells into the vascular wall and 26 prevented atherosclerosis development in mice. This study aimed to investigate the effect of curcumin 27 on monocyte adhesion and transendothelial migration (TEM) and to decipher the underlying 28 mechanisms of these actions.

29 Human umbilical vein endothelial cells (HUVECs) were exposed to curcumin (0.5-1 µM) for 3 hours 30 prior to their activation by Tumor Necrosis Factor alpha (TNF-α). Endothelial permeability, monocyte 31 adhesion and transendothelial migration assays were conducted under static condition and shear stress 32 that mimics blood flow. We further investigated the impact of curcumin on signaling pathways and on 33 the expression of genes using macroarrays.

34 Pre-exposure of endothelial cells to curcumin reduced monocyte adhesion and their transendothelial 35 migration in both static and shear stress conditions. Curcumin also prevented changes in both 36 endothelial permeability and the area of HUVECs when induced by TNF-α. We showed that curcumin 37 modulated the expression of 15 genes involved in the control of cytoskeleton and endothelial junction 38 dynamic. Finally, we showed that curcumin inhibited NF-κB signaling likely through an antagonist 39 interplay with several kinases as suggested by molecular docking analysis.

40 Our findings demonstrate the ability of curcumin to reduce monocyte TEM through a multimodal 41 regulation of the endothelial cell dynamics with a potential benefit 42 on the vascular endothelial function barrier.

43

44

45 **Keywords ;**

46 Curcumin

47 Endothelial cell

48 Endothelial permeability

49 Monocyte adhesion
<figure><img src="image_3.png" /></figure>
## 51 INTRODUCTION

Impairment of the endothelial cell function is a major factor in the development of vascular damage. Endothelial cell dysfunction is characterized by the abnormal expression of genes including those coding for adhesion molecules, which leads to the firm adhesion of leukocytes to the endothelium and their subsequent infiltration into the vascular wall, which is one of the initial events in atherosclerosis onset [1].

Curcumin is a polyphenolic compound of turmeric derived from the rhizome of *Curcuma longa*. This compound is commonly used as a dietary spice and food coloring agent. Curcumin exhibits potent antioxidant and anti-inflammatory properties that could be beneficial for vascular health [2]. Some clinical data showed that daily curcumin intake, from 450 mg/day to 8 g/day for 1 to 4 months, can reduce some cardiovascular risk factors such as systolic blood pressure [3], serum triglycerides and low density lipoprotein (LDL) [4], and can improve endothelial function [5]. Moreover, curcumin has been shown to reduce the development of atheromatous lesions in different animal models of atherosclerosis [6-11]. Coban et al. revealed a decrease in macrophages in the atherosclerotic lesions of ApoE/- mice that were supplemented with dietary curcumin [6]. In the same study, the authors identified that most of the differentially expressed genes in response to curcumin supplementation were entailed in leukocyte recruitment and transmigration. Um et al. showed a lower expression of endothelial adhesion molecules (VCAM-1, ICAM-1) in the aortas of rabbits fed with a high cholesterol diet supplemented with curcumin [9].

*In vitro* studies have demonstrated that curcumin reduces monocyte adhesion to human endothelial cells activated by the pro-inflammatory tumor necrosis factor alpha (TNF-α) by modulating the expression of adhesion molecules [6, 12-15]. Down-regulation of such molecules has been related to an inhibitory effect of curcumin on several cell-signaling molecules, of which NF-κB is one of the major transcription factors activated by TNF-α [16, 17].

Following their firm adhesion to the endothelium, monocytes can migrate through the endothelium. This process, also known as diapedesis, transendothelial migration (TEM) or extravasation, is a dynamic process entailing endothelial cytoskeleton remodeling and disruption of
78 endothelial cell junctions that cause an increase in vascular permeability [18, 19]. These cellular
79 modifications can be induced *in vitro* by the stimulation of endothelial cells with TNF-α [20, 21]. So
80 far, little is known about the effect of curcumin on the regulation of cytoskeleton and junctional
81 dynamics in the endothelium. It has been reported that oral supplementation of rats with curcumin may
82 reduce coronary artery permeability to a fluorescent macromolecule [22]. To our knowledge, no
83 studies have investigated the impact of curcumin on leukocyte transmigration through the
84 endothelium.

85 Based on the above considerations, this study aimed to investigate the effect of curcumin on
86 monocyte transendothelial migration and on the expression of genes involved in this process. Special
87 attention was paid to reproduce the physiological environment of vascular endothelial cells as closely
88 as possible. For that, the cells were exposed to low concentrations of curcumin for a 3-hour period, in
89 respect to pharmacokinetics data of curcumin [23]. Moreover, effects of curcumin were also
90 investigated in endothelial cells exposed to physiological shear stress that mimicked blood flow.
91

## 2. MATERIALS AND METHODS

### Chemicals

95 Curcumin was purchased from Extrasynthese (France). Reagents for cell culture (RPMI 1640,
96 penicillin/streptomycin, Trypsin/EDTA, Foetal Bovine Serum) were purchased from PAN biotech
97 (Germany). Gelatin, paraformaldehyde (PFA), FITC-dextran, dimethylsulfoxide (DMSO), anti-
98 protease cocktail, anti-phosphatase cocktail, Nonidet P40 and DAPI were obtained from Sigma-
99 Aldrich (France). Recombinant-human-TNF-α and MCP-1 were obtained from R&D systems and
100 Miltenyi Biotec SAS (Paris, France) respectively.

### Cell Culture

103 Human umbilical vein endothelial cells (HUVECs) (Lonza, Amboise, France) were cultured in a
104 phenol-red-free endothelial growth medium supplemented with 2% fetal bovine serum, 0.4% human
105 fibroblast growth factor, 0.1% human epidermal growth factor, 0.1% vascular endothelial growth
factor, 0.1% insulin-like growth factor, 0.1% ascorbic acid, 0.1% heparin, 0.1% gentamicin/amphotericin-B and 0.04% hydrocortisone (all from Lonza, France). A monocyte THP1 cell line obtained from ACC (Manassas, VA) was cultured in RPMI 1640 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Both cultures were maintained at 37°C in humidified atmosphere with 5% CO₂.

111

## 112 Cell adhesion assay

HUVECs were seeded into 0.2% gelatin-coated 24-well plates at a density of 50,000 cells/well. At 60-70% confluence, cells were made quiescent for 24 hours in medium with 0.2% FBS, and then were exposed to 0.5 µM, 1 µM curcumin or a DMSO vehicle (0.02%) for 3 hours. Following pre-incubation with curcumin, inflammatory stress was induced by 1 ng/ml of TNF-α for 4 hours. After TNF-α stimulation, 5x10⁵ THP1 cells were added to each well, and cells were co-incubated for 15 minutes. Non-adhering THP1 cells were rinsed away by washing with 1X DPBS. The remaining attached cells were subsequently detached using trypsin/EDTA (0.05%/0.02%), fixed with 1% PFA and stored at 4°C prior to immunostaining and analysis by flow cytometry.

121

## 122 Flow cytometry

Cell immunostaining was performed according to the manufacturer instructions (Miltenyi Biotec SAS, Paris, France). Briefly, fixed cells from the adhesion assay were pelleted by centrifugation at 300 g for 10 minutes and treated with FcR blocking reagent for 10 minutes. Subsequently, they were stained with CD45 antibody conjugated to FITC for 30 minutes in the dark. IgG2a isotype antibodies conjugated to FITC were used as a negative control. The number of CD45-positive cells (THP1) and unstained cells (HUVECs) was measured by BD FACSCalibur (BD Bioscience, San Jose, CA), set at 20,000 events, and analyzed using CellQuest Pro software, version 5.1.1 (BD Bioscience, San Jose, CA) for Macintosh. Relative monocyte adhesion to endothelial cells was expressed as the ratio of monocytes to HUVECs.

132

## 133 Monocyte transendothelial migration assay
134

HUVECs were seeded at 15,000 cells/well onto Corning Transwell filters (5 µm pore, 6.5 mm diameter) (Corning, USA) previously coated with 0.2% gelatin and grown to confluence. Cells were made quiescent for 24 hours in medium with 0.2% FBS and were then exposed to 0.5 µM and 1 µM curcumin or vehicle, respectively (DMSO, 0.02%), for 3 hours. Following this incubation, 100 ng/mL of MCP-1 were added to the lower chamber, and 5x10⁵ THP1 above HUVECs. Cells were incubated for 3 hours at 37°C and 5% CO₂. THP1 cells that had transmigrated to the lower chamber were harvested and counted using a Beckman cell coulter (Beckman Coulter, USA). The results are expressed as a percentage of transmigrated monocytes relative to the condition ‘vehicle + TNF-α’.

135

136

137

138

139

140

141

142

143

## Endothelium permeability assay

144

145

146

147

148

149

150

151

As for monocyte transendothelial migration assay, confluent cultured HUVECs loaded onto Corning Transwell filters were made quiescent for 24 hours in medium with 0.2% FBS before being exposed to 0.5 µM or 1 µM curcumin or vehicle (0.02% DMSO) for 3 hours, followed by stimulation with 1 ng/ml of TNF-α for 4 hours. Permeability of the endothelium was evaluated by passage of FITC-dextran at 40 kDa through endothelial monolayer. One hundred microliters of FITC-dextran at 40 kDa (2 mg/mL) was added to the upper chamber and allowed to equilibrate for 20 minutes, after which FITC fluorescence (excitation 488 nm; emission 520 nm) in the lower chamber was measured using a Biotek synergy 2 plate reader (BioTek, France). Four independent experiments were performed.

152

153

## Endothelial cell area measurement

154

155

156

157

158

159

160

161

HUVECs were seeded on 0.2% gelatin-coated glass coverslips (13 mm diameter). At 60-70% of confluence, HUVECs were treated with TNF-α as previously described for the monocyte adhesion assay. After 15 min of THP1-HUVECs co-incubation, cells were rinsed to remove non-adherent cells and then fixed with 1% PFA for 15 min at 4°C. Fixed cells were permeabilized by a 1% Triton X100/1X PBS/3% BSA solution, and the actin cytoskeleton was stained with rhodamine-phalloidin (Invitrogen). Nuclei were counterstained with DAPI (100 nM). Fluorescence was detected and imaged using a Zeiss AxioPlan E microscope (Karl Zeiss, Jena, Germany) and a Zeiss AxioCam MRM (Carl Zeiss Vision, Hallbergmoos, Germany). The mean endothelial cell area was measured in 10 fields of
162

view per condition from 2 independent experiments using “ImageJ” image analysis software
(http://www.imagej.nih.gov/).

163

164

165

Chemoattractant capacity of TNF-α activated HUVECs

166

167

168

169

170

171

172

173

Chemoattractant potency of HUVECs was evaluated using a transwell assay and conditioned media harvested from HUVECs exposed 3 hours to 1 µM curcumin or DMSO vehicle, with or without stimulation by 1 ng/mL TNF-α for 4 hours. Conditioned media were added to the lower chamber and 5x10⁵ THP1 was added above the filter. After incubation for 3 hours at 37°C with 5% CO₂, transmigrated THP1 cells were harvested and counted using a Beckman cell counter (Beckman Coulter, USA). The results were expressed as a percentage of transmigrated monocytes compared to that measured in response to the supplementation of basal conditioned medium with 100 ng/mL MCP-1.

174

175

Monocyte adhesion and transendothelial migration assessment under shear stress

176

177

178

179

180

181

182

183

184

185

186

187

188

HUVECs were seeded at 15,000 cells/cm² in gelatin-coated microchannels of the Vena8 Endothelial Biochip (Cellix, Ireland). After adhesion (2 hours), biochips were connected to the Kima pump (Cellix), and cells were grown under a 1 Hz-pulsatile aortic shear stress of 7 dyne/cm² for 72 h. Cells were exposed to low concentrations of curcumin (0.5-1 µM) or a vehicle (DMSO 0.02%) for 3 hours prior to their stimulation with 1 ng/ml of TNF-α for 4 hours under shear stress. Following TNF-α stimulation, a suspension of 2x10⁶ THP1/mL was perfused through the microchannels at 0.5 dyne/cm² for 10 min. Interactions between monocytes and HUVECs were visualized with videomicroscopy using phase contrast optics (Diaphot-TMD microscope and DS-Fi 1 CCD camera, Nikon, USA). Each experimental condition was recorded on a single field for the 10 min minutes. Additionally, images of ten fields were captured for each condition and distributed along the channel. Firmly adherent monocytes to HUVECs accumulated throughout the experiment, and those that had transmigrated were quantified at the end of the experiment. Adherent and transmigrated monocytes were identified based on their phase white and round appearance and on their phase dark and flattened shape,
189

respectively [24]. Quantifications were performed on pictures obtained from 2-3 independent experiments using “ImageJ” image analysis software (www.imagej.nih.gov).

191

192

Quantitative real-time PCR analysis

193

Total RNAs were extracted using Trizol Reagent (Thermo Fisher Scientific, France) following manufacturer conditions. RNA concentration was measured using a spectrophotometer NanoDrap ND-1000 (LabTech, Ringmer, UK) and their quality checked by 1% agarose gel electrophoresis. 1ug total RNA extract was used as template for single-strand cDNA synthesis using High Capacity cDNA RT kit (Applied Biosystems, USA) in a total volume of 20 µl containing 1 X RT buffer, 4 mM dNTP mix, 1 X random primers, 50 U reverse transcriptase and 20 U RNase inhibitor. The reverse transcription reactions were ran under the following conditions: 25°C for 10 min, 37°C for 120 min and 85°C for 5 s. PCR was carried out in the final volume of 20 µl containing 10 µl Power SYBR Green PCR Master Mix kit (Applied Biosystems, USA), 0.4 µl of each primer at 10 pmol/µl and 2 µl of the cDNA solution. The primers used were: h-MCP-1-F: TCA TAG CAG CCA CCT TCA TTC C, h-MCP-1-R: GGA CAC TTG CTG CTG GTG ATT C; GAPDH-F: CTG GGC TAC ACT GAG CAC C, GAPDH-R: AAG TGG TCG TTG AGG GCA ATG. RT-PCR amplification was performed using an Applied Biosystems Prism 7900HT (Applied Biosystems, France) system with the following thermal cycler conditions: 2 min at 50°C and 10 min at 94.5°C, followed by 40 cycles of 30 s at 97°C and 30 s at 59.7°C. Raw data were analyzed using Sequence Detection System (SDS) Software v2.4 (Applied Biosystems, Courtaboeuf, France) and compared by the ΔΔCt method. Results are expressed relative to the housekeeping gene transcript quantity and normalized to untreated cells.

194

195

196

197

198

199

200

201

202

203

204

205

206

207

208

209

210

211

212

213

214

215

216

Impact of curcumin on the expression of 93 other genes in HUVECs involved in endothelial cell function was performed using TaqMan Low Density Array (TLDA) (Applied Biosystem). Genes were chosen according to their role in atherosclerosis development from published articles (supplemental table S1). Two hundreds ng (2 µl) cDNA of each sample was combined with, 98 µl of nuclease-free water and 100 µl 2X TaqMan Universal PCR Master Mix (Applied Biosystems, USA) for the quantitative real-time PCR (qPCR) measurements. This mixture was divided equally over two sample-loading ports of the TLDA. The arrays were centrifuged once (1 min, 1300 rpm at room temperature)
217 to equally distribute the sample over the wells. Subsequently, the card was sealed to prevent an
218 exchange between wells. qPCR amplification was performed using an Applied Biosystems Prism
219 7900HT system (Applied Biosystem, USA) with the following thermal cycler conditions: 2 min at
220 50°C and 10 min at 94.5°C, followed by 40 cycles of 30 s at 97°C and 30 s at 59.7°C. Raw data were
221 analyzed using Sequence Detection System (SDS) Software v2.4 (Applied Biosystems). Analyses
222 were performed in triplicates. Bioinformatics analyzes with these genes were performed using the
223 miRWalk database (link) to predict the validated miRNAs modulated by curcumin.

224

225 **Western blot analyses**

226 HUVECs pre-exposed to curcumin or vehicle were harvested at the end of a 4-hour-TNF-α activation
227 period and washed twice with ice-cold PBS. Total proteins were extracted using lysis buffer
228 containing 50 mmol/L Tris pH 7.8, 150 mmol/L NaCl, 0.5% sodium deoxycholate, 1% NP40, anti-
229 protease and anti-phosphatase. Protein concentration was determined using BCA protein assay reagent
230 Kit (Interchem). Lysates were loaded onto a 10% SDS-polyacrylamide gel for electrophoresis and then
231 transferred onto immobilon-P membrane (GE Healthcare). The membrane was incubated in 5%
232 (wt/vol) dried milk protein in TBS containing 0.05% Tween-20 for 1 hour, and then further reacted
233 with primary antibodies: rabbit anti-iCAM (Santa Cruz, 1:1000), rabbit anti-vCAM-1 (1:1000, GTX),
234 rabbit anti-NFκB total (1:1000, Cell Signaling), rabbit anti-NFκB phosphor-ser536 (1:1000, Cell
235 Signaling), rabbit anti-IκBα total (1:1000, Cell Signaling) and rabbit anti-IκBα phosphor-ser32
236 (1:1000, Cell Signaling). After extensive washes, membrane was incubated with anti-rabbit IgG
237 antibody conjugated to HRP (1:5000, Santa Cruz, USA). Protein bands were visualized using ECL
238 detection kit (Millipore, USA) and then analyzed using Image J software (www.imagej.nih.gov).
239 Graphs represent protein level expressed as the mean +/- standard deviation of 4 independent
240 experiments.

241

242 **Molecular docking analysis**
Prior to virtual screening (docking), an academic license of *MarvinSketch* was used for drawing, displaying of 2D structure and 3D optimization of curcumin and generation of the required input file for docking (Tripos MOL2 file format), MarvinSketch 16.10.24.0, 2016, ChemAxon (https://www.chemaxon.com). Docking also requires the 3D structure of the target and it was necessary to cross-reference 2 on-line databases in order to identify the possible targets and their high-resolution complete 3D structures – a resolution better than 2.0 Å being recommended for docking [25]: The Universal Protein Resource – UniProt (http://www.uniprot.org) and RCSB Protein Data Bank – PDB (http://www.rcsb.org). Since our search criteria did not had positive feed-back, we constructed homologues models with the help of SWISS-MODEL[26]. Virtual screening of curcumin against the selected targets was conducted with *PyRx* – Python Prescription 0.9.2 using AutoDock Vina [27] as docking software. AutoDock Vina uses a scoring function inspired by X-score and tuned with PDBbind dataset [28] to predict the noncovalent binding of macromolecules and automatically calculates the grid maps and clusters the results. Data extraction and rendering of docking poses were performed with Molegro Molecular Viewer 2.5 (MMV – Molegro, A CLC bio company, Aarhus N, Denmark). The "backface culling" method was used for simulation of the molecular surface of proteins. This method allowed us to determine whether a polygon of a graphical object is visible (e.g., primary structure, secondary structure, or inner part of binding side shown as a molecular surface); when it was not visible, the polygon was "culled" from rendering process, which increases efficiency by reducing the number of polygons the hardware has to draw [29]. All docking runs were conducted in an extended search space, set to cover the entire target, with a volume bigger than 27.0 Å³ and the exhaustiveness was manually increased to 80in order to improve the accuracy of predictions.

264

## Statistical analyses

Prism software, version 6.0.c (GraphPad, La Jolla, CA) for Macintosh, was used for the statistical analysis of data. Data were analyzed using one-way analysis of variance (ANOVA) and Dunnett's multiple comparison test. For the analysis of gene expression data, two-way ANOVA was performed, followed by Dunnett's multiple comparison test with a single pooled variance. A value of p < 0.05 was considered significant.

243

244

245

246

247

248

249

250

251

252

253

254

255

256

257

258

259

260

261

262

263

264

265

266

267

268

269

270
271

272

273
274

## 3. Results

275

276

### Curcumin decreases both monocyte adhesion to endothelial cells and their transendothelial migration (TEM)

277

278

As expected, activation of HUVECs by TNF-α stimulated the firm adhesion of monocytes to the endothelial surface compared to those incubated with the non-stimulated HUVECs (3-fold increase; p<0.001) (fig. 1A). This adhesion was significantly reduced by 25% (p<0.001) in HUVECs pre-exposed to curcumin at 0.5 µM and 1 µM compared to vehicle-treated endothelial cells.

279

280

281

282

Regarding TEM, fig. 1B shows that the chemoattractant protein MCP-1 induced a 4.5-fold higher monocyte migration in a transwell assay compared to the cells incubated without MCP-1 (p<0.001).

283

284

The magnitude of this MCP-1-induced migration was significantly reduced when HUVECs were pre-treated with 1 µM curcumin (-24%, p<0.05).

285

286

287

### Curcumin preserves endothelium permeability and cell surface area during inflammation

288

289

Cell permeability assays, performed using Transwell system, revealed that stimulation of HUVECs by TNF-α resulted in a significant 4-fold increase of endothelial permeability to FITC-dextran (fig. 2A).

290

291

This increase in permeability was inhibited by 34% with a pre-incubation of endothelial cells with 1 µM curcumin (p=0.028). In addition, the endothelial cell surface within the endothelium cell monolayer was modified in response to TNF-α induced monocyte adhesion to HUVECs (fig. 2B). A 50% decrease of the mean cell area of HUVECs was observed after 1 hour of monocyte/TNF-α-activated endothelial cells interaction (p<0.01). In TNF-α activated HUVECs pre-exposed to 1 µM curcumin, the surface of HUVECs decreased by only 25%. Thus, exposure of endothelial cells to 1 µM curcumin reduced by 50% the TNF-α-induced contraction of HUVECs (p<0.05).

292

293

294

295

296

297

298

### Curcumin affects neither chemoattractive capacity of TNF-α-activated endothelial cells nor the expression of MCP-1

299

300

Fig. 3A shows that monocyte chemotaxis induced by the culture medium from TNF-α-activated
endothelial cells was similar to the chemotaxis induced by the chemokine MCP-1. In addition, we observed that the treatment of endothelial cells with 1 µM curcumin did not affect the endothelial mediated monocyte chemotaxis. The expression of the gene coding for MCP-1 in endothelial cells was quantified by RT-qPCR (fig. 3B). A 4-hour treatment of HUVECs with TNF-α highly increased the expression of this gene (p<0.01). Pre-exposure of endothelial cells to curcumin before their activation by TNF-α had no significant effect on the TNF-α-induced expression of the MCP-1 gene.

307

### 308 Curcumin decreases monocyte-endothelial interaction under shear stress

Under physiological shear stress (~7 dyne/cm²), TNF-α significantly increased the tight adhesion of monocytes to endothelial cells by 20-fold (Fig. 4A and suppl. Movie). The pretreatment of HUVECs by 1 µM curcumin significantly reduced monocyte adhesion by 36% (p=0.017). A tendency for a decrease was observed with 0.5 µM of curcumin but did not reach significance.

Time-lapse microscopy assessing monocyte transendothelial migration under shear stress showed that the pretreatment of HUVECs with 0.5 µM or 1 µM curcumin tended to decrease the number of migrated monocytes in comparison to migration observed for the vehicle-treated cells under shear stress (Fig. 4B and suppl. Movie).

317

### 318 Curcumin modulates expression of genes in HUVECs

Of 93 genes for which the expression of TEM was evaluated, seventeen were identified as not expressed in our condition (fig. 5A and suppl. Table 1). Our nutrigenomic study revealed that the exposure of endothelial cells to curcumin prior their activation by TNF-α significantly modulated the expression of sixteen genes. Bioinformatic analyses showed that these differentially expressed genes are involved in different cellular processes, such as antioxidant defense (SOD-1), metabolism (IGFR1), cell signaling (CAV1, CASK, PDPK1), focal adhesion (PXN, VCL, ADAM10), intercellular junction (TJP2) and cytoskeleton organization (CDC42 subunits, ARPC2, PAK4, ACTN1, VIM). For three genes, tendencies in the modulation of their expression were observed; these genes encode two
327

small Rho GTPases involved in the regulation of the cytoskeleton (RHOC and RAC1) and for the
CALD1 gene which encodes caldesmon 1.

328

329

Additionally, bioinformatic analysis, performed using the miRWalk database, identified 459 putative
miRNAs potentially modulated by curcumin and involved in the regulation of the aforementioned
genes differentially expressed (data not shown).

330

331

332

Moreover, in TNF-α non-stimulated endothelial cells, a low basal expression of cell adhesion proteins (VCAM-1 and ICAM-1) was observed (fig. 5B6 A-C). A 4-hour treatment of endothelial cells with TNF-α highly increased the expression of these two proteins. The expression of these proteins was not affected by the exposure of HUVECs to curcumin prior to TNF-α-activation.

333

334

335

336

337

### Curcumin modulates phosphorylation of NF-κB cell-signaling proteins

338

We observed that a 3-hour exposure to 0.5 µM or 1 µM of curcumin in TNF-α non-stimulated endothelial cells did not modify the basal phosphorylation level of NF-κB p65 and IκBα (fig. 6).

339

340

Following stimulation with TNF-α, levels of phosphorylated NF-κB p65 (ser536) and phosphorylated IκBα (ser32) were increased, and the total amount of IκBα was reduced compared to those observed in the non-stimulated endothelial cells. As a result, the phosphorylated/total ratio of NF-κB p65 and IκBα were higher in TNF-α-activated cells compared to the non-stimulated cells. Pre-exposure of HUVECs to 0.5 µM curcumin prior to their stimulation to TNF-α did not modify the phosphorylated/total ratio of NF-κB p65 and IκBα. In contrast, the exposure to 1 µM curcumin significantly reduced the level of phosphorylation of NF-κB p65 (fig. 6 A and D) without affecting that of IκBα (fig. 6 A and E).

341

342

343

344

345

346

347

348

349

### Curcumin reveals favorable binding properties to cell-signaling proteins

350

The analysis of protein binding between curcumin and cell-signaling proteins of the NF-κB signaling pathway revealed that of the 13 proteins evaluated (Table 1 & suppl. Table 2), 2 protein structures were not available, and 7 computational predictions had a low degree of confidence due to low resolution of the 3D structure of the target (resolution > 2.0 Å is recommended for docking) [25]. In

351

352

353
silico predictions demonstrated that curcumin could interact directly with TAK1 and PDK1 to form a covalent bond within the active site of these proteins with a binding affinity of -7.8 Kcal/mol and -6.5 Kcal/mol, respectively (Table 1). Curcumin can also interplay with the kinases AKT 1 and AKT 2 through a non-covalent interaction of -7.3 kCal/mol and -6.3 kCal/mol, respectively. These interactions can be seen in fig. 7.

## 4. Discussion

In this study, we set out to identify the cellular effects and the molecular mechanisms responsible for the ability of curcumin to prevent impairments in endothelial cell function. A special focus was on the impact of curcumin on endothelial cell processes involved in the interactions between vascular endothelium and circulating immune cells.

The bioavailability of pure curcumin is known to be very low in humans, with plasma concentrations most often not detectable for administered dose below 4 grams and peaking at 1.77 µM after the intake of 8 g of curcumin [30-33]. However, in recent years many strategies have been developed to markedly increase the bioavailability of oral curcumin, including the use of natural enhancers or of encapsulation systems to improve its delivery [34], with a positive impact on the bio-efficacy of curcumin in clinical studies [35]. With such approaches, the concentrations of curcumin tested in the present study (0.5 and 1 µM) could be easily reached in the plasma of human subjects consuming a reasonable dose of curcumin. Our study showed a reduction of monocyte adhesion to endothelial cells exposed to curcumin (0.5 µM and 1 µM) for 3 hours that corresponds to the resident time of curcumin within blood circulation after curcumin intake [23]. This effect did not seem to result from an altered monocyte chemotaxis as revealed by the lack of effect of curcumin on chemoattractive potency of activated endothelial cells. This result is not consistent with previous studies reporting that curcumin can alter the expression of the gene coding for chemoattractant chemokine (MCP-1) in several cell lines [36]. However, these studies used curcumin at much higher concentrations than those used in the present study.
To mimic the endothelial dysfunction observed in vascular diseases [21], endothelial cells were activated by the inflammatory cytokine TNF-α. This activation induces the expression of cell adhesion molecules such as ICAM and VCAM, which are involved in the firm adhesion of monocytes [20]. In our *in vitro* conditions, curcumin at 1 µM significantly reduced monocyte adhesion, but it did not modify the expression of adhesion molecules induced by TNF-α. This observation is not in line with results from other studies showing a reduction of monocyte adhesion to endothelial cells along with a decrease in the expression of adhesion molecules. This discrepancy could result from differences in the experimental conditions used, for example, in previous studies, endothelial cells were treated with higher concentrations of curcumin (≥ 5 µM) for 1 hour prior to TNF-α activation [12-14]. Nevertheless, the chronic consumption of curcumin has been associated *in vivo* with a decrease in the expression of genes coding for ICAM-1 and VCAM-1 in the aorta of atherosclerotic rabbits [9]. Tethering of monocytes with endothelial cells activates ICAM-1 and VCAM-1 clustering in lipid raft domain of the endothelial membrane [37-39] resulting in the firm adhesion of monocytes to the endothelial cells prior to their extravasation [39-41]. Wójciak-Stothard et al. [42] have demonstrated that inhibition of adhesion molecule clustering reduced the adhesion of monocytes to activated endothelial cells. Moreover, it has been reported that curcumin, at 5 to 10 µM, may affect delocalization of integrin and the formation of a signaling complex at a lipid raft, both of which are required for cell mobility [43, 44]. Taken together, these results suggest that the decrease in monocyte adhesion to endothelial cells could result from the capacity of curcumin to affect the subcellular movement of adhesion molecules required for monocyte adhesion and diapedesis.

Following the firm adhesion to endothelial cells, monocytes undergo diapedesis. For the first time, we showed that curcumin can also reduce monocyte transendothelial migration. Extravasation of monocytes across the endothelium requires regulation of endothelial junctional permeability [18]. In addition, we demonstrated that 1 µM curcumin reduced the TNF-α-induced increase in permeability of the confluent monolayer of HUVECs. In agreement with these results, Li et al. previously reported that oral supplementation with curcumin protects against the alteration in permeability of the coronary artery as measured in a rat model of coronary disease [22]. Moreover, in western-diet induced
atherosclerotic mice, curcumin has been previously shown to reduce intestinal epithelium permeability by acting on tight junction proteins involved in the selective barrier function such ZO-1 and claudin 1 [10, 18]. Endothelial permeability is controlled not only by the regulation of cell-cell junctions but also by cytoskeleton dynamics [45]. Indeed, re-organization of actin into contractile stress fibers or microtubule disassembly increases endothelial contraction, resulting in interendothelial gap formation and consequently an increase in permeability. Interestingly, we observed that endothelial cells pre-exposed to curcumin were less sensitive to TNF-α induced modification of the cell surface, which is dependent on the actin cytoskeleton. All together, these results suggest that curcumin may have an impact on endothelial permeability by acting on the regulation of junction proteins and on the dynamics of the actin cytoskeleton. This hypothesis is in agreement with the lower number of monocytes within the atheromatous lesion observed in curcumin-supplemented ApoE/- mice [6].

It is worth noting that endothelial cells in vessels are exposed to the luminal surface of blood flow that induces a physical strain called shear stress [46, 47]. This physiological shear stress is known to activate endothelial mechanoreceptors resulting in the induction of the expression of transcription factors such as Krüppel-like factor 2 (KLP2) and nuclear factor (erythroid-derived 2)-like 2 (NRF2), among others [48]. The activation of these factors contributes to the preservation of endothelial function by inducing vasodilatory, antioxidant, antithrombotic, antiadherent and antiangiogenic responses. Here, we demonstrated for the first time that the exposure of endothelial cells to low concentrations of curcumin under physiological shear stress could reduce monocyte adhesion and extravasation. To the best of our knowledge, only one other study, that of Chacko et al., demonstrated a similar observation under flow in response to 1 µM of isoflavone, another subfamily of polyphenols [49, 50]. In addition, we observed that the magnitude of the effect of curcumin on adhesion and TEM in the condition of shear stress was markedly higher (+44% and +37%, respectively) than those observed in the static condition. Hence, one can speculate that curcumin may potentiate the beneficial effect of physiological shear stress on endothelial function.

To decipher the underlying mechanisms of action of curcumin, we also screened for the expression of 93 genes coding for adhesion and permeability proteins in response to curcumin exposure in endothelial cells for the first time. Among the genes significantly modulated by the
exposure to curcumin, our results revealed the overexpression of PXN, VIM and VCL, coding for paxilin, vimentin and vinculin, respectively, which are three proteins involved in the focal adhesion of endothelial cells to extracellular matrix [19]. Down-regulation of such proteins was associated to an increase in endothelial barrier permeability [48, 51]. Similarly, the expression of genes TJP2 and CASK, coding the tight junction protein 2 (also called ZO-2) and the Ca²⁺/Calmodulin dependent serine protein kinase (CASK), both involved in endothelial cell-cell junction [45], were shown to increase in response to curcumin. Such change in the expression of these genes has been described to be associated to a reduction in endothelial permeability [52]. Our gene expression assay also showed that curcumin affects the expression of caveolin-1 (CAV1), which plays an important role in the organization of protein interactions between cells and the extracellular matrix that contributes to control endothelial permeability [53]. Moreover, we observed that curcumin modulated the expression of two Rho GTPases (ARPC2 and CDC42), which are known to control microtubule dynamics and actin-rich surface projections involved in TEM process [54], and the expression of the PDPK1 kinase, which regulates cell migration [55]. In addition, we observed the modulation of PAK4 expression by curcumin. PAK4 has been described to activate the downstream target RHOA involved in the regulation of the assembly of focal adhesions and actin stress fibers [19]. The expression of caldesmon (CALD1), a binding protein that plays a role in the stabilization of actin filament structure [56], was also significantly upregulated in curcumin-exposed HUVECs. Taken together, the changes in gene expression that we observed suggest that curcumin could maintain endothelium integrity by reinforcing cell-cell and cell-matrix interactions, and by stabilizing endothelial cell cytoskeleton through nutrigenomic effect. This hypothesis fits with the observed impact of curcumin on endothelial cell function and surface area.

The molecular mechanisms of curcumin involve a large range of targets including signaling pathways and transcription factors [16]. In a previous animal study, we demonstrated that a dietary supplementation with 0.2% curcumin inhibits atherosclerosis by affecting NF-κB-transcriptional activity [6]. In the present *in vitro* study, we have stimulated endothelial cells with TNF-α, an activator of the NF-κB signaling pathway [57], and we showed that 1 µM of curcumin significantly
464

reduced the phosphorylation of NF-κB p65 (serine 536). Others have previously described such an
inhibition with curcumin in a similar range of concentrations [12, 13, 21]. In contrast to previous
observations [12-14], in our experimental conditions, inhibition of NF-κB by curcumin did not result
in a lowering of the expression of NF-κB target genes such as ICAM and VCAM. However, Cobb et
al. [58] reported that induction of VCAM1 can be NF-κB-independent and that proteasome can
regulate VCAM-1 and ICAM-1 expression in endothelial cells without affecting NF-κB activation.
Recently, Hasima et al. [59] suggested that curcumin can modulate proteasomal pathways in a dose-
dependent manner. Thus, it can be speculated that curcumin may regulate the expression of adhesion
molecules in a different way than the NF-κB pathway, potentially by acting on proteasomal activity.

465

466

467

468

469

470

471

472

473

The inhibition of the NF-κB pathway observed in response to exposure of endothelial cells to curcumin is also supported by our results obtained *in silico* that revealed favorable binding of curcumin to some kinases, namely, TAK1, PDK1 and AKT. TAK1 and PDK1 have been reported to activate the NF-κB canonical pathway by phosphorylating the IκB Kinase β (IKKβ) [60]. AKT is known to be activated by TNF-α stimulation [61] and to regulate the activity of NF-κB by inducing phosphorylation and subsequent degradation of inhibitor of NF-κB (IκB) [62]. Regarding the binding mode of curcumin in the active domain of these kinases, the occupancy and the interactions we observed mimic those of a heterocyclic core of known inhibitors of kinases that have been shown to attenuate NF-κB activation [63-65]. Similarly, Gupta et al. reported that curcumin might interact directly with a large range of targets including signaling proteins and protein kinases, leading in their inhibition [66]. In addition, *in silico* structural evaluation highlighted that quercetin, another polyphenol, may interact and therefore inhibit serine/threonine kinases such as AKT [67]. Collectively, our *in silico* analysis suggests that curcumin can bind to cell-signaling proteins and affect their activity, which consequently results in modulation of the expression of genes as we observed.

474

475

476

477

478

479

480

481

482

483

484

485

486

487

Another possible mechanism of action by which curcumin could modulate adhesion and
monocyte transendothelial migration in endothelial cells may involve the modulation of miRNAs.
miRNA are noncoding single-strand RNAs of 22 nucleotides that constitute a class of gene regulators
at the posttranscriptional level [68]. Among the miRNAs potentially modulated by curcumin and

488

489

490
involved in the regulation of the 15 aforementioned genes shown as differentially expressed in response to curcumin in the present study, 4 were previously detected as modulated by dietary curcumin in murine ApoE⁻/⁻ liver (miR-137, miR-188-5p, miR-324-5p and miR-335-5p) [69]. MiR-137 has been described as a regulator of Cdc42 expression and a Rho GTPase [70], and miR-335-5p is involved in cytoskeleton dynamics [71]. In addition, Howell et al. have recently described the ability of curcumin to modulate 29 miRNAs in epithelial cell line ARPE-19 [72]. Nine of these miRNAs were reported to regulate the expression of cell-cell junction proteins and adhesion molecules (miR181a, miR18a, miR150 miR101, miR126, miR181b, miR27a, miR155, and miR20A) [73]. These findings were supported by bioinformatic analyses suggesting that the miRNAs detected as modulated by curcumin in murine ApoE⁻/⁻ liver [69] and in murine melanoma [74] may regulate several pathways influencing cell communication and focal adhesion. Therefore, further investigation is needed to determine the impact of curcumin on miRNAs as post-transcriptional mode of regulation of monocyte TEM in endothelial cells.

In conclusion, this *in vitro* study provides new mechanistic insights into the ability of dietary curcumin to preserve vascular health during inflammation, as summarized in fig. 8. Our findings clearly support that, in physiologically relevant conditions, exposure of endothelial cells to curcumin can reduce endothelial permeability and monocyte adhesion and transmigration, ultimately promoting vascular integrity. This effect seems to be related to a complex and multimodal action of curcumin on the intracellular dynamics of endothelial cells. However, these mechanisms warrant further investigation to be fully understood.
512 **Statement of authorship**

513 The authors' responsibilities were as follows: LEM designed methods and experiments, carried out the
514 experiments, analyzed the data and wrote the paper; SM carried out cell experiments and flow
515 cytometry analysis, and DB performed the gene expression assay; RT performed molecular docking
516 analysis; NBC participated in discussion and revision of the article; DM & CM carried out study
517 design, data interpretation and manuscript preparation. All authors have contributed to read and
518 approved the manuscript.

519

520

521 **Conflict of interest**

522 None of the authors has any conflict of interest to declare.

523

524 **Acknowledgements**

525 We acknowledge networking support by the COST Action FA1403-POSITIVe (Interindividual
526 variation in response to consumption of plant food bioactives and determinants involved).

527
528

529

Legends

530

531

**Figure 1: Effect of curcumin on monocyte adhesion to endothelial cells and their transendothelial migration (TEM) in the static condition of culture.** (A) Monocyte adhesion to TNF-α activated HUVECs after a 3-hour exposure of endothelial cells to curcumin. In the absence of curcumin, HUVECs were exposed to vehicle (0.2% DMSO). The results are expressed as a percentage of monocyte adhesion to ‘vehicle+TNF-α’ treated HUVECs that is normalized at 100%. (B) TEM of monocytes in response to MCP-1 across HUVEC monolayer exposed to curcumin or vehicle for 3 hours. The results are expressed as the percentage of migration measured in the vehicle+MCP-1 condition that is normalized at 100%. Bars represent the mean +/- SEM of 3-4 independent experiments. Significances: \*p\le0.05, \*\*p\le0.01, \*\*\*p\le0.001.

532

533

534

535

536

537

538

539

540

541

**Figure 2: Effect of curcumin on endothelium integrity.** (A) Permeability of the HUVEC monolayer to FITC-dextran (40 kDa) after 3-hour exposure to vehicle or curcumin (0.5 and 1 µM) followed by 4-hour TNF-α activation. The results are expressed as a percentage of permeability measured in ‘vehicle+TNF-α’ treated HUVECs that is normalized at 100% (n=4). (B, C) Impact of curcumin exposition on mean HUVEC surface area measured after monocyte/TNF-α-activated endothelial cells interaction. (B) Representative immunostainings of the actin cytoskeleton of HUVECs after 3-hour exposure to vehicle or curcumin (1 µM) followed by 4-hour TNF-α activation and co-incubation with monocytes (Magnitude: 20x). (C) Quantification of the mean HUVEC surface area. The results are expressed in comparison to the mean area measured at steady state (n=3). Significances: \*p\le0.05, \*\*p\le0.001.

542

543

544

545

546

547

548

549

550

551

552

**Figure 3: Chemoattractive potency of TNF-α-activated endothelial cells in response to curcumin.** (A) Transmigrated monocytes stimulated by the conditioned media harvested from vehicle/-TNF-α-, vehicle/+TNF-α- and Cur 1 µM/+TNF-α-HUVECs were assessed using a transwell

553

554
assay. Bars represent means +/- SEM (n=4). (B) Relative expression of gene coding the chemoattractive protein MCP-1 normalized first to the expression of the housekeeping gene GAPDH and then to the relative expression detected in vehicle/+TNF-α-treated HUVECs. Values indicate fold-changes expressed as the mean +/- SEM (n=4). Significances: *p≤0.05, **p≤0.01, ***p≤0.001.

**Figure 4: Effect of curcumin on monocyte adhesion to endothelial cells and their transendothelial migration under shear stress.** (A) Monocyte adhesion to TNF-α activated HUVECs after a 3-hour exposure of endothelial cells to curcumin under 1 Hz pulsatile and 7 dyne/cm² shear stress. The results are expressed as a percentage of monocyte adhesion to ‘vehicle/+TNF-α’ treated HUVECs that normalized at 100%. (B) Transmigrated monocytes accumulated throughout the entire 10-minute duration of the adhesion assay. The results are expressed as a percentage of transmigrated monocytes to ‘vehicle/+TNF-α’ treated HUVECs that is normalized at 100%. Significances: *p≤0.05, **p≤0.01, ***p≤0.001.

**Figure 5: Gene expression in curcumin-exposed HUVECs.** (A) Expression of 93 genes coding proteins involving TEM quantified by TLDA. Values (mean +/- SEM, n=4) indicate gene expression fold change measured in 1 µM Cur/+TNF-α HUVECs compared to Vehicle/+TNF-α-exposed HUVECs. (B) Expression of endothelial adhesion molecules (ICAM and VCAM) at protein level. *p≤0.05 in comparison to vehicle/-TNF-α group.

**Figure 6: Impact of curcumin on endothelial adhesion molecules and on NF-κB signaling pathway proteins in HUVECs.** (A) Representative images of a western blot showing the detection of adhesion molecules, -total NF-κB and phosphor-ser536, total IκBα and phosphor-ser532, and GAPDH in HUVECs exposed to curcumin (0.5 µM and 1 µM) or vehicle and stimulated by 1 ng/mL TNF-α for 4h (vCAM and ICAM) or 15 min (NF-κB and IκBα). (B, C) Quantification of the expression of endothelial adhesion molecules (vCAM and ICAM respectively). (D, E) Ratios of phosphorylated/total NF-κB and IκBα respectively. protein are expressed as the means Graphs represent means +/- SEM (n=3). *p≤0.05.
582
583 Figure 7: 3D structures from *in silico* docking of curcumin to cell proteins of the NF-κB signaling pathway.
584
585

586
587 Figure 8: Overview of the demonstrated and putative mechanisms by which curcumin can modulate endothelial cell activation.
588
589 Table 1: Binding score of curcumin interplay with cell-signaling proteins. (N/A: none

590
591
592
593 Table 1: Binding score of curcumin interplay with cell-signaling proteins. (N/A: none
adapted)

593
594 Supplementary data

594
595
596
597
598
599
600
601
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
618
619
620
621
622
623
624
625
626
627
628
629
630
631
632
633
634
635
636
637
638
639
640
641
642
643
644
645
646
647
648
649
650
651
652
653
654
655
656
657
658
659
660
661
662
663
664
665
666
667
668
669
670
671
672
673
674
675
676
677
678
679
680
681
682
683
684
685
686
687
688
689
690
691
692
693
694
695
696
697
698
699
700
701
702
703
704
705
706
707
708
709
710
711
712
713
714
715
716
717
718
719
720
721
722
723
724
725
726
727
728
729
730
731
732
733
734
735
736
737
738
739
740
741
742
743
744
745
746
747
748
749
750
751
752
753
754
755
756
757
758
759
760
761
762
763
764
765
766
767
768
769
770
771
772
773
774
775
776
777
778
779
780
781
782
783
784
785
786
787
788
789
790
791
792
793
794
795
796
797
798
799
800
801
802
803
804
805
806
807
808
809
810
811
812
813
814
815
816
817
818
819
820
821
822
823
824
825
826
827
828
829
830
831
832
833
834
835
836
837
838
839
840
841
842
843
844
845
846
847
848
849
850
851
852
853
854
855
856
857
858
859
860
861
862
863
864
865
866
867
868
869
870
871
872
873
874
875
876
877
878
879
880
881
882
883
884
885
886
887
888
889
890
891
892
893
894
895
896
897
898
899
900
901
902
903
904
905
906
907
908
909
910
911
912
913
914
915
916
917
918
919
920
921
922
923
924
925
926
927
928
929
930
931
932
933
934
935
936
937
938
939
940
941
942
943
944
945
946
947
948
949
950
951
952
953
954
955
956
957
958
959
960
961
962
963
964
965
966
967
968
969
970
971
972
973
974
975
976
977
978
979
980
981
982
983
984
985
986
987
988
989
990
991
992
993
994
995
996
997
998
999
1000

**Suppl. Movie 1:** Monocyte rolling, tethering, adhesion and transmigration on TNF-α activated HUVECs under physiological shear stress.

594
595
596
597
598
599
600
601
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
618
619
620
621
622
623
624
625
626
627
628
629
630
631
632
633
634
635
636
637
638
639
640
641
642
643
644
645
646
647
648
649
650
651
652
653
654
655
656
657
658
659
660
661
662
663
664
665
666
667
668
669
670
671
672
673
674
675
676
677
678
679
680
681
682
683
684
685
686
687
688
689
690
691
692
693
694
695
696
697
698
699
700
701
702
703
704
705
706
707
708
709
710
711
712
713
714
715
716
717
718
719
720
721
722
723
724
725
726
727
728
729
730
731
732
733
734
735
736
737
738
739
740
741
742
743
744
745
746
747
748
749
750
751
752
753
754
755
756
757
758
759
760
761
762
763
764
765
766
767
768
769
770
771
772
773
774
775
776
777
778
779
780
781
782
783
784
785
786
787
788
789
790
791
792
793
794
795
796
797
798
799
800
801
802
803
804
805
806
807
808
809
810
811
812
813
814
815
816
817
818
819
820
821
822
823
824
825
826
827
828
829
830
831
832
833
834
835
836
837
838
839
840
841
842
843
844
845
846
847
848
849
850
851
852
853
854
855
856
857
858
859
860
861
862
863
864
865
866
867
868
869
870
871
872
873
874
875
876
877
878
879
880
881
882
883
884
885
886
887
888
889
890
891
892
893
894
895
896
897
898
899
900
901
902
903
904
905
906
907
908
909
910
911
912
913
914
915
916
917
918
919
920
921
922
923
924
925
926
927
928
929
930
931
932
933
934
935
936
937
938
939
940
941
942
943
944
945
946
947
948
949
950
951
952
953
954
955
956
957
958
959
960
961
962
963
964
965
966
967
968
969
970
971
972
973
974
975
976
977
978
979
980
981
982
983
984
985
986
987
988
989
990
991
992
993
994
995
996
997
998
999
1000

596
597
598
599
600
601
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
618
619
620
621
622
623
624
625
626
627
628
629
630
631
632
633
634
635
636
637
638
639
640
641
642
643
644
645
646
647
648
649
650
651
652
653
654
655
656
657
658
659
660
661
662
663
664
665
666
667
668
669
670
671
672
673
674
675
676
677
678
679
680
681
682
683
684
685
686
687
688
689
690
691
692
693
694
695
696
697
698
699
700
701
702
703
704
705
706
707
708
709
710
711
712
713
714
715
716
717
718
719
720
721
722
723
724
725
726
727
728
729
730
731
732
733
734
735
736
737
738
739
740
741
742
743
744
745
746
747
748
749
750
751
752
753
754
755
756
757
758
759
760
761
762
763
764
765
766
767
768
769
770
771
772
773
774
775
776
777
778
779
780
781
782
783
784
785
786
787
788
789
790
791
792
793
794
795
796
797
798
799
800
801
802
803
804
805
806
807
808
809
810
811
812
813
814
815
816
817
818
819
820
821
822
823
824
825
826
827
828
829
830
831
832
833
834
835
836
837
838
839
840
841
842
843
844
845
846
847
848
849
850
851
852
853
854
855
856
857
858
859
860
861
862
863
864
865
866
867
868
869
870
871
872
873
874
875
876
877
878
879
880
881
882
883
884
885
886
887
888
889
890
891
892
893
894
895
896
897
898
899
900
901
902
903
904
905
906
907
908
909
910
911
912
913
914
915
916
917
918
919
920
921
922
923
924
925
926
927
928
929
930
931
932
933
934
935
936
937
938
939
940
941
942
943
944
945
946
947
948
949
950
951
952
953
954
955
956
957
958
959
960
961
962
963
964
965
966
967
968
969
970
971
972
973
974
975
976
977
978
979
980
981
982
983
984
985
986
987
988
989
990
991
992
993
994
995
996
997
998
999
1000

600
601
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
618
619
620
621
622
623
624
625
626
627
628
629
630
631
632
633
634
635
636
637
638
639
640
641
642
643
644
645
646
647
648
649
650
651
652
653
654
655
656
657
658
659
660
661
662
663
664
665
666
667
668
669
670
671
672
673
674
675
676
677
678
679
680
681
682
683
684
685
686
687
688
689
690
691
692
693
694
695
696
697
698
699
700
701
702
703
704
705
706
707
708
709
710
711
712
713
714
715
716
717
718
719
720
721
722
723
724
725
726
727
728
729
730
731
732
733
734
735
736
737
738
739
740
741
742
743
744
745
746
747
748
749
750
751
752
753
754
755
756
757
758
759
760
761
762
763
764
765
766
767
768
769
770
771
772
773
774
775
776
777
778
779
780
781
782
783
784
785
786
787
788
789
790
791
792
793
794
795
796
797
798
799
800
801
802
803
804
805
806
807
808
809
810
811
812
813
814
815
816
817
818
819
820
821
822
823
824
825
826
827
828
829
830
831
832
833
834
835
836
837
838
839
840
841
842
843
844
845
846
847
848
849
850
851
852
853
854
855
856
857
858
859
860
861
862
863
864
865
866
867
868
869
870
871
872
873
874
875
876
877
878
879
880
881
882
883
884
885
886
887
888
889
890
891
892
893
894
895
896
897
898
899
900
901
902
903
904
905
906
907
908
909
910
911
912
913
914
915
916
917
918
919
920
921
922
923
924
925
926
927
928
929
930
931
932
933
934
935
936
937
938
939
940
941
942
943
944
945
946
947
948
949
950
951
952
953
954
955
956
957
958
959
960
961
962
963
964
965
966
967
968
969
970
971
972
973
974
975
976
977
978
979
980
981
982
983
984
985
986
987
988
989
990
991
992
993
994
995
996
997
998
999
1000

600
601
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
618
619
620
621
622
623
624
625
626
627
628
629
630
631
632
633
634
635
636
637
638
639
640
641
642
643
644
645
646
647
648
649
650
651
652
653
654
655
656
657
658
659
660
661
662
663
664
665
666
667
668
669
670
671
672
673
674
675
676
677
678
679
680
681
682
683
684
685
686
687
688
689
690
691
692
693
694
695
696
697
698
699
700
701
702
703
704
705
706
707
708
709
710
711
712
713
714
715
716
717
718
719
720
721
722
723
724
725
726
727
728
729
730
731
732
733
734
735
736
737
738
739
740
741
742
743
744
745
746
747
748
749
750
751
752
753
754
755
756
757
758
759
760
761
762
763
764
765
766
767
768
769
770
771
772
773
774
775
776
777
778
779
780
781
782
783
784
785
786
787
788
789
790
791
792
793
794
795
796
797
798
799
800
801
802
803
804
805
806
807
808
809
810
811
812
813
814
815
816
817
818
819
820
821
822
823
824
825
826
827
828
829
830
831
832
833
834
835
836
837
838
839
840
841
842
843
844
845
846
847
848
849
850
851
852
853
854
855
856
857
858
859
860
861
862
863
864
865
866
867
868
869
870
871
872
873
874
875
876
877
878
879
880
881
882
883
884
885
886
887
888
889
890
891
892
893
894
895
896
897
898
899
900
901
902
903
904
905
906
907
908
909
910
911
912
913
914
915
916
917
918
919
920
921
922
923
924
925
926
927
928
929
930
931
932
933
934
935
936
937
938
939
940
941
942
943
944
945
946
947
948
949
950
951
952
953
954
955
956
957
958
959
960
961
962
963
964
965
966
967
968
969
970
971
972
973
974
975
976
977
978
979
980
981
982
983
984
985
986
987
988
989
990
991
992
993
994
995
996
997
998
999
1000
605 [1] S. Sitia, L. Tomasoni, F. Atzeni, G. Ambrosio, C. Cordiano, A. Catapano, S. Tramontana, F.
606 Perticone, P. Naccarato, P. Camici, E. Picano, L. Cortigiani, M. Bevilacqua, L. Milazzo, D. Cusi, C.
607 Barlassina, P. Sarzi-Puttini, M. Turiel, From endothelial dysfunction to atherosclerosis, Autoimmunity
608 reviews 9(12) (2010) 830-4.

609 [2] A.S. Strimpakos, R.A. Sharma, Curcumin: preventive and therapeutic properties in laboratory
610 studies and clinical trials, Antioxidants & redox signaling 10(3) (2008) 511-45.

611 [3] P. Khajehdehi, B. Zanjaninejad, E. Aflaki, M. Nazarinia, F. Azad, L. Malekmakan, G.R.
612 Dehghanzadeh, Oral supplementation of turmeric decreases proteinuria, hematuria, and systolic
613 blood pressure in patients suffering from relapsing or refractory lupus nephritis: a randomized and
614 placebo-controlled study, Journal of renal nutrition : the official journal of the Council on Renal
615 Nutrition of the National Kidney Foundation 22(1) (2012) 50-7.

616 [4] S. Chuengsamarn, S. Rattanamongkolgul, B. Phonrat, R. Tungtrongchitr, S. Jirawatnotai, Reduction
617 of atherogenic risk in patients with type 2 diabetes by curcuminoid extract: a randomized controlled
618 trial, The Journal of nutritional biochemistry 25(2) (2014) 144-50.

619 [5] H. Nakayama, N. Tsuge, H. Sawada, N. Masamura, S. Yamada, S. Satomi, Y. Higashi, A single
620 consumption of curry improved postprandial endothelial function in healthy male subjects: a
621 randomized, controlled crossover trial, Nutrition journal 13 (2014) 67.

622 [6] D. Coban, D. Milenkovic, A. Chanet, J. Khallou-Laschet, L. Sabbe, A. Palagani, W. Vanden Berghe,
623 A. Mazur, C. Morand, Dietary curcumin inhibits atherosclerosis by affecting the expression of genes
624 involved in leukocyte adhesion and transendothelial migration, Molecular nutrition & food research
625 56(8) (2012) 1270-81.

626 [7] J.F. Zhao, L.C. Ching, Y.C. Huang, C.Y. Chen, A.N. Chiang, Y.R. Kou, S.K. Shyue, T.S. Lee, Molecular
627 mechanism of curcumin on the suppression of cholesterol accumulation in macrophage foam cells
628 and atherosclerosis, Molecular nutrition & food research 56(5) (2012) 691-701.

629 [8] S.T. Hasan, J.M. Zingg, P. Kwan, T. Noble, D. Smith, M. Meydani, Curcumin modulation of high fat
630 diet-induced atherosclerosis and steatohepatosis in LDL receptor deficient mice, Atherosclerosis
631 232(1) (2014) 40-51.

632 [9] M.Y. Um, K.H. Hwang, W.H. Choi, J. Ahn, C.H. Jung, T.Y. Ha, Curcumin attenuates adhesion
633 molecules and matrix metalloproteinase expression in hypercholesterolemic rabbits, Nutr Res 34(10)
634 (2014) 886-93.

635 [10] S.S. Ghosh, J. Bie, J. Wang, S. Ghosh, Oral supplementation with non-absorbable antibiotics or
636 curcumin attenuates western diet-induced atherosclerosis and glucose intolerance in LDLR-/- mice--
637 role of intestinal permeability and macrophage activation, PloS one 9(9) (2014) e108577.

638 [11] Q. Hao, X. Chen, X. Wang, B. Dong, C. Yang, Curcumin Attenuates Angiotensin II-Induced
639 Abdominal Aortic Aneurysm by Inhibition of Inflammatory Response and ERK Signaling Pathways,
640 Evidence-based complementary and alternative medicine : eCAM 2014 (2014) 270930.

641 [12] Y.S. Kim, Y. Ahn, M.H. Hong, S.Y. Joo, K.H. Kim, I.S. Sohn, H.W. Park, Y.J. Hong, J.H. Kim, W. Kim,
642 M.H. Jeong, J.G. Cho, J.C. Park, J.C. Kang, Curcumin attenuates inflammatory responses of TNF-alpha-
643 stimulated human endothelial cells, Journal of cardiovascular pharmacology 50(1) (2007) 41-9.

644 [13] A. Kumar, S. Dhawan, N.J. Hardegen, B.B. Aggarwal, Curcumin (Diferuloylmethane) inhibition of
645 tumor necrosis factor (TNF)-mediated adhesion of monocytes to endothelial cells by suppression of
646 cell surface expression of adhesion molecules and of nuclear factor-kappaB activation, Biochemical
647 pharmacology 55(6) (1998) 775-83.

648 [14] H.S. Lee, M.J. Lee, H. Kim, S.K. Choi, J.E. Kim, H.I. Moon, W.H. Park, Curcumin inhibits TNFalpha-
649 induced lectin-like oxidised LDL receptor-1 (LOX-1) expression and suppresses the inflammatory
650 response in human umbilical vein endothelial cells (HUVECs) by an antioxidant mechanism, Journal of
651 enzyme inhibition and medicinal chemistry 25(5) (2010) 720-9.
652

653

654

[15] R. Olszanecki, A. Gebska, R. Korbut, The role of haem oxygenase-1 in the decrease of endothelial intercellular adhesion molecule-1 expression by curcumin, Basic & clinical pharmacology & toxicology 101(6) (2007) 411-5.

655

656

[16] S. Shishodia, Molecular mechanisms of curcumin action: gene expression, Biofactors 39(1) (2013) 37-55.

657

658

[17] S.C. Gupta, B. Sung, J.H. Kim, S. Prasad, S. Li, B.B. Aggarwal, Multitargeting by turmeric, the golden spice: From kitchen to clinic, Molecular nutrition & food research 57(9) (2013) 1510-28.

659

660

[18] A.E. Daniel, J.D. van Buul, Endothelial junction regulation: a prerequisite for leukocytes crossing the vessel wall, Journal of innate immunity 5(4) (2013) 324-35.

661

662

[19] J.T. Parsons, A.R. Horwitz, M.A. Schwartz, Cell adhesion: integrating cytoskeletal dynamics and cellular tension, Nature reviews. Molecular cell biology 11(9) (2010) 633-43.

663

664

665

[20] E. VandenBerg, M.D. Reid, J.D. Edwards, H.W. Davis, The role of the cytoskeleton in cellular adhesion molecule expression in tumor necrosis factor-stimulated endothelial cells, Journal of cellular biochemistry 91(5) (2004) 926-37.

666

667

668

669

[21] L. Laviola, M.R. Orlando, M.A. Incalza, C. Caccioppoli, M. Melchiorre, A. Leonardini, A. Cignarelli, F. Tortosa, R. Labarbuta, S. Martemucci, C. Pacelli, T. Cocco, S. Perrini, A. Natalicchio, F. Giorgino, TNFalpha signals via p66(Shc) to induce E-Selectin, promote leukocyte transmigration and enhance permeability in human endothelial cells, PloS one 8(12) (2013) e81930.

670

671

672

[22] X. Li, Y. Lu, Y. Sun, Q. Zhang, Effect of curcumin on permeability of coronary artery and expression of related proteins in rat coronary atherosclerosis heart disease model, International journal of clinical and experimental pathology 8(6) (2015) 7247-53.

673

674

675

[23] M. Heger, R.F. van Golen, M. Broekgaarden, M.C. Michel, The molecular basis for the pharmacokinetics and pharmacodynamics of curcumin and its metabolites in relation to cancer, Pharmacological reviews 66(1) (2014) 222-307.

676

677

[24] C.W. Smith, Endothelial adhesion molecules and their role in inflammation, Canadian journal of physiology and pharmacology 71(1) (1993) 76-87.

678

[25] Chemoinformatics Approaches to Virtual Screening Royal Society of chemistry2008.

679

680

681

682

[26] M. Biasini, S. Bienert, A. Waterhouse, K. Arnold, G. Studer, T. Schmidt, F. Kiefer, T. Gallo Cassarino, M. Bertoni, L. Bordoli, T. Schwede, SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information, Nucleic acids research 42(Web Server issue) (2014) W252-8.

683

684

685

[27] O. Trott, A.J. Olson, AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading, Journal of computational chemistry 31(2) (2010) 455-61.

686

687

[28] R. Wang, X. Fang, Y. Lu, C.Y. Yang, S. Wang, The PDBbind database: methodologies and updates, Journal of medicinal chemistry 48(12) (2005) 4111-9.

688

689

[29] C.-Y. Han, Y.-H. Im, L.-S. Kim, Geometry Engine Architecture with Early Backface Culling Hardware, Computers & Graphics 29(3) (2005) 415-425.

690

691

692

693

[30] A.L. Cheng, C.H. Hsu, J.K. Lin, M.M. Hsu, Y.F. Ho, T.S. Shen, J.Y. Ko, J.T. Lin, B.R. Lin, W. Ming-Shiang, H.S. Yu, S.H. Jee, G.S. Chen, T.M. Chen, C.A. Chen, M.K. Lai, Y.S. Pu, M.H. Pan, Y.J. Wang, C.C. Tsai, C.Y. Hsieh, Phase I clinical trial of curcumin, a chemopreventive agent, in patients with high-risk or pre-malignant lesions, Anticancer research 21(4B) (2001) 2895-900.

694

695

696

697

[31] R.A. Sharma, S.A. Euden, S.L. Platton, D.N. Cooke, A. Shafayat, H.R. Hewitt, T.H. Marczylo, B. Morgan, D. Hemingway, S.M. Plummer, M. Pirmohamed, A.J. Gescher, W.P. Steward, Phase I clinical trial of oral curcumin: biomarkers of systemic activity and compliance, Clinical cancer research : an official journal of the American Association for Cancer Research 10(20) (2004) 6847-54.

698

699

700

701

[32] N. Dhillon, B.B. Aggarwal, R.A. Newman, R.A. Wolff, A.B. Kunnumakkara, J.L. Abbruzzese, C.S. Ng, V. Badmaev, R. Kurzrock, Phase II trial of curcumin in patients with advanced pancreatic cancer, Clinical cancer research : an official journal of the American Association for Cancer Research 14(14) (2008) 4491-9.
702

703

704

[33] R.E. Carroll, R.V. Benya, D.K. Turgeon, S. Vareed, M. Neuman, L. Rodriguez, M. Kakarala, P.M. Carpenter, C. McLaren, F.L. Meyskens, Jr., D.E. Brenner, Phase IIa clinical trial of curcumin for the prevention of colorectal neoplasia, Cancer Prev Res (Phila) 4(3) (2011) 354-64.

705

706

[34] G. Shoba, D. Joy, T. Joseph, M. Majeed, R. Rajendran, P.S. Srinivas, Influence of piperine on the pharmacokinetics of curcumin in animals and human volunteers, Planta medica 64(4) (1998) 353-6.

707

708

709

[35] H. Mirzaei, A. Shakeri, B. Rashidi, A. Jalili, Z. Banikazemi, A. Sahebkar, Phytosomal curcumin: A review of pharmacokinetic, experimental and clinical studies, Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie 85 (2017) 102-112.

710

711

[36] M.S. Karimian, M. Pirro, M. Majeed, A. Sahebkar, Curcumin as a natural regulator of monocyte chemoattractant protein-1, Cytokine & growth factor reviews (2016).

712

713

[37] R.W. Tilghman, R.L. Hoover, E-selectin and ICAM-1 are incorporated into detergent-insoluble membrane domains following clustering in endothelial cells, FEBS letters 525(1-3) (2002) 83-7.

714

715

716

717

[38] O. Barreiro, M. Yanez-Mo, J.M. Serrador, M.C. Montoya, M. Vicente-Manzanares, R. Tejedor, H. Furthmayr, F. Sanchez-Madrid, Dynamic interaction of VCAM-1 and ICAM-1 with moesin and ezrin in a novel endothelial docking structure for adherent leukocytes, The Journal of cell biology 157(7) (2002) 1233-45.

718

719

720

[39] J.M. van Gils, J.J. Zwaginga, P.L. Hordijk, Molecular and functional interactions among monocytes, platelets, and endothelial cells and their relevance for cardiovascular diseases, Journal of leukocyte biology 85(2) (2009) 195-204.

721

722

723

[40] J.D. van Buul, J. van Rijssel, F.P. van Alphen, M. Hoogenboezem, S. Tol, K.A. Hoeben, J. van Marle, E.P. Mul, P.L. Hordijk, Inside-out regulation of ICAM-1 dynamics in TNF-alpha-activated endothelium, PloS one 5(6) (2010) e11336.

724

725

726

[41] J.D. van Buul, J. van Rijssel, F.P. van Alphen, A.M. van Stalborch, E.P. Mul, P.L. Hordijk, ICAM-1 clustering on endothelial cells recruits VCAM-1, Journal of biomedicine & biotechnology 2010 (2010) 120328.

727

728

729

[42] B. Wojciak-Stothard, L. Williams, A.J. Ridley, Monocyte adhesion and spreading on human endothelial cells is dependent on Rho-regulated receptor clustering, The Journal of cell biology 145(6) (1999) 1293-307.

730

731

732

[43] M.L. Lin, Y.C. Lu, H.Y. Chen, C.C. Lee, J.G. Chung, S.S. Chen, Suppressing the formation of lipid raft-associated Rac1/PI3K/Akt signaling complexes by curcumin inhibits SDF-1alpha-induced invasion of human esophageal carcinoma cells, Molecular carcinogenesis 53(5) (2014) 360-79.

733

734

735

[44] Y.H. Soung, J. Chung, Curcumin inhibition of the functional interaction between integrin alpha6beta4 and the epidermal growth factor receptor, Molecular cancer therapeutics 10(5) (2011) 883-91.

736

737

[45] E. Vandenbroucke, D. Mehta, R. Minshall, A.B. Malik, Regulation of endothelial junctional permeability, Annals of the New York Academy of Sciences 1123 (2008) 134-45.

738

739

[46] A.M. Malek, S.L. Alper, S. Izumo, Hemodynamic shear stress and its role in atherosclerosis, Jama 282(21) (1999) 2035-42.

740

741

742

743

[47] Y.S. Chatzizisis, A.U. Coskun, M. Jonas, E.R. Edelman, C.L. Feldman, P.H. Stone, Role of endothelial shear stress in the natural history of coronary atherosclerosis and vascular remodeling: molecular, cellular, and vascular behavior, Journal of the American College of Cardiology 49(25) (2007) 2379-93.

744

745

[48] D.A. Chistiakov, A.N. Orekhov, Y.V. Bobryshev, Effects of shear stress on endothelial cells: go with the flow, Acta Physiol (Oxf) (2016).

746

747

748

[49] B.K. Chacko, R.T. Chandler, T.L. D'Alessandro, A. Mundhekar, N.K. Khoo, N. Botting, S. Barnes, R.P. Patel, Anti-inflammatory effects of isoflavones are dependent on flow and human endothelial cell PPARgamma, The Journal of nutrition 137(2) (2007) 351-6.

749

750

751

752

[50] B.K. Chacko, R.T. Chandler, A. Mundhekar, N. Khoo, H.M. Pruitt, D.F. Kucik, D.A. Parks, C.G. Kevil, S. Barnes, R.P. Patel, Revealing anti-inflammatory mechanisms of soy isoflavones by flow: modulation of leukocyte-endothelial cell interactions, American journal of physiology. Heart and circulatory physiology 289(2) (2005) H908-15.
753

[51] P. Fu, P.V. Usatyuk, J. Jacobson, A.E. Cress, J.G. Garcia, R. Salgia, V. Natarajan, Role played by paxillin and paxillin tyrosine phosphorylation in hepatocyte growth factor/sphingosine-1-phosphate-mediated reactive oxygen species generation, lamellipodia formation, and endothelial barrier function, Pulmonary circulation 5(4) (2015) 619-30.

754

755

756

757

758

759

[52] S.W. Lee, W.J. Kim, H.O. Jun, Y.K. Choi, K.W. Kim, Angiopoietin-1 reduces vascular endothelial growth factor-induced brain endothelial permeability via upregulation of ZO-2, International journal of molecular medicine 23(2) (2009) 279-84.

760

761

[53] P.G. Frank, S.E. Woodman, D.S. Park, M.P. Lisanti, Caveolin, caveolae, and endothelial cell function, Arteriosclerosis, thrombosis, and vascular biology 23(7) (2003) 1161-8.

762

763

[54] N. Heemskerk, J. van Rijssel, J.D. van Buul, Rho-GTPase signaling in leukocyte extravasation: an endothelial point of view, Cell adhesion & migration 8(2) (2014) 67-75.

764

765

[55] L. Primo, L. di Blasio, C. Roca, S. Droetto, R. Piva, B. Schaffhausen, F. Bussolino, Essential role of PDK1 in regulating endothelial cell migration, The Journal of cell biology 176(7) (2007) 1035-47.

766

767

768

[56] T. Mirzapoiazova, I.A. Kolosova, L. Romer, J.G. Garcia, A.D. Verin, The role of caldesmon in the regulation of endothelial cytoskeleton and migration, Journal of cellular physiology 203(3) (2005) 520-8.

769

770

[57] T. Lawrence, The nuclear factor NF-kappaB pathway in inflammation, Cold Spring Harbor perspectives in biology 1(6) (2009) a001651.

771

772

773

[58] R.R. Cobb, K.A. Felts, G.C. Parry, N. Mackman, Proteasome inhibitors block VCAM-1 and ICAM-1 gene expression in endothelial cells without affecting nuclear translocation of nuclear factor-kappa B, European journal of immunology 26(4) (1996) 839-45.

774

775

[59] N. Hasima, B.B. Aggarwal, Targeting proteasomal pathways by dietary curcumin for cancer prevention and treatment, Current medicinal chemistry 21(14) (2014) 1583-94.

776

777

[60] L.A. Madge, J.S. Pober, TNF signaling in vascular endothelial cells, Experimental and molecular pathology 70(3) (2001) 317-25.

778

779

780

781

[61] K. Murao, T. Ohyama, H. Imachi, T. Ishida, W.M. Cao, H. Namihira, M. Sato, N.C. Wong, J. Takahara, TNF-alpha stimulation of MCP-1 expression is mediated by the Akt/PKB signal transduction pathway in vascular endothelial cells, Biochemical and biophysical research communications 276(2) (2000) 791-6.

782

783

784

[62] D. Bai, L. Ueno, P.K. Vogt, Akt-mediated regulation of NFkappaB and the essentialness of NFkappaB for the oncogenicity of PI3K and Akt, International journal of cancer 125(12) (2009) 2863-70.

785

786

787

788

[63] T. Muraoka, M. Ide, K. Morikami, M. Irie, M. Nakamura, T. Miura, T. Kamikawa, M. Nishihara, H. Kashiwagi, Discovery of a potent and highly selective transforming growth factor beta receptor-associated kinase 1 (TAK1) inhibitor by structure based drug design (SBDD), Bioorganic & medicinal chemistry 24(18) (2016) 4206-17.

789

790

791

792

[64] T. Hayashi, J.H. Mo, X. Gong, C. Rossetto, A. Jang, L. Beck, G.I. Elliott, I. Kufareva, R. Abagyan, D.H. Broide, J. Lee, E. Raz, 3-Hydroxyanthranilic acid inhibits PDK1 activation and suppresses experimental asthma by inducing T cell apoptosis, Proceedings of the National Academy of Sciences of the United States of America 104(47) (2007) 18619-24.

793

794

795

[65] Z. Song, X. Zhu, R. Jin, C. Wang, J. Yan, Q. Zheng, A. Nanda, D.N. Granger, G. Li, Roles of the kinase TAK1 in CD40-mediated effects on vascular oxidative stress and neointima formation after vascular injury, PloS one 9(7) (2014) e101671.

796

797

798

[66] S.C. Gupta, S. Prasad, J.H. Kim, S. Patchva, L.J. Webb, I.K. Priyadarsini, B.B. Aggarwal, Multitargeting by curcumin as revealed by molecular interaction studies, Natural product reports 28(12) (2011) 1937-55.

799

800

801

[67] B. Baby, P. Antony, W. Al Halabi, Z. Al Homedi, R. Vijayan, Structural insights into the polypharmacological activity of quercetin on serine/threonine kinases, Drug design, development and therapy 10 (2016) 3109-3123.

802

803

[68] D.P. Bartel, MicroRNAs: genomics, biogenesis, mechanism, and function, Cell 116(2) (2004) 281-97.
[69] D. Milenkovic, B. Jude, C. Morand, miRNA as molecular target of polyphenols underlying their biological effects, Free radical biology & medicine 64 (2013) 40-51.

[70] M. Liu, N. Lang, M. Qiu, F. Xu, Q. Li, Q. Tang, J. Chen, X. Chen, S. Zhang, Z. Liu, J. Zhou, Y. Zhu, Y. Deng, Y. Zheng, F. Bi, miR-137 targets Cdc42 expression, induces cell cycle G1 arrest and inhibits invasion in colorectal cancer cells, International journal of cancer 128(6) (2011) 1269-79.

[71] X.S. Cui, S.C. Sun, Y.K. Kang, N.H. Kim, Involvement of microRNA-335-5p in cytoskeleton dynamics in mouse oocytes, Reproduction, fertility, and development 25(4) (2013) 691-9.

[72] J.C. Howell, E. Chun, A.N. Farrell, E.Y. Hur, C.M. Caroti, P.M. Iuvone, R. Haque, Global microRNA expression profiling: curcumin (diferuloylmethane) alters oxidative stress-responsive microRNAs in human ARPE-19 cells, Molecular vision 19 (2013) 544-60.

[73] Y. Zhuang, H. Peng, V. Mastej, W. Chen, MicroRNA Regulation of Endothelial Junction Proteins and Clinical Consequence, Mediators of Inflammation vol.2016 (2016).

[74] I.N. Dahmke, C. Backes, J. Rudzitis-Auth, M.W. Laschke, P. Leidinger, M.D. Menger, E. Meese, U. Mahlknecht, Curcumin intake affects miRNA signature in murine melanoma with mmu-miR-205-5p most significantly altered, PloS one 8(12) (2013) e81122.
<table><caption>Table 1</caption><thead><tr><th>Target</th><th>Binding Affinity of curcumin (Kcal/mol)</th><th>Number of H-Bonds</th><th>Location of H-Bonds</th></tr></thead><tbody><tr><td>PDK1</td><td>-6,5</td><td>1</td><td>Tyr126 of the active site</td></tr><tr><td>AKT1</td><td>-7.3</td><td>0</td><td>N/A</td></tr><tr><td>AKT2</td><td>-6.3</td><td>0</td><td>N/A</td></tr><tr><td>TAK1</td><td>-7.8</td><td>1</td><td>Lys227 (coil) of the protein kinase domain, blocking the access at the ATP binding site and interaction with MAPK8IP1</td></tr></tbody></table>
<figure><img src="image_4.png" /><figcaption>Figure 1</figcaption></figure>

<figure><img src="image_5.png" /></figure>
<figure><img src="image_6.png" /><figcaption>Figure 2</figcaption></figure>
<caption>Figure 3</caption>

A

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /><figcaption>Figure 4</figcaption></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>
<figure><img src="image_11.png" /><figcaption>Figure 5</figcaption></figure>
<figure><img src="image_12.png" /><figcaption>Figure 6</figcaption></figure>
<figure><img src="image_13.png" /><figcaption>Figure 7
Click here to download high resolution image</figcaption></figure>
<figure><img src="image_14.png" /><figcaption>Figure8
Click here to download high resolution image</figcaption></figure>