6-Shogaol was purchased from PhytoLab GmbH & Co. KG (Vestenbergsgreuth, Germany) and 6-gingerol from MedChemExpress (Monmouth Junction, NJ, United States) and was used in the experiments in different concentrations in a range between 1 and 100 µM. The concentration used in each experiment is stated in the respective figure. Human recombinant interleukin 1β (IL-1β), tumor necrosis factor (TNF), stromal cell-derived factor 1 (SDF-1), vascular endothelial growth factor (VEGF₁₆₅), and murine recombinant VEGF₁₆₅ were obtained from PeproTech (Rocky Hill, NJ, United States). Lipopolysaccharide (LPS) from Escherichia coli O127:B8 was from Sigma-Aldrich (St. Louis, MO, United States). The concentrations of 6-shogaol for the treatment of endothelial cells were chosen up to the maximum concentration without inducing cytotoxicity. The concentration of 0.1 up to 150 µM was previously used for other cell types (Bischoff-Kont and Fürst, 2021). For 6-gingerol, the same concentration range was used. Moreover, a study employing HUVECs for the analysis of effects of 6-gingerol on microvessel-like structure formation used concentrations of up to 30 µM (Zhog et al., 2019). The concentration of 10 ng/ml of TNF is an established hyperphysiological concentration that is frequently used in order to obtain a strong inflammatory induction in HUVECs (Stark et al., 2019; Krishnatas et al., 2021). For IL-1β, 5 ng/ml was found to be a reasonable concentration to induce the inflammatory response in endothelial cells and is within the commonly applied concentration range (Kihara et al., 2021). The applied concentration of human VEGF₁₆₅ for the in vitro assays resulted in appropriate angiogenic induction and is commonly used for angiogenesis-related experiments in endothelial cells (Liu et al., 2009; Bräutigam et al., 2019; Bischoff-Kont et al., 2021). Murine VEGF₁₆₅ was applied according to the publication of Baker et al. (2011). LPS was applied in order to induce an appropriate inflammatory response in HUVECs according to the concentrations recommended in the literature (Zeuke et al., 2002). The applied concentration of SDF-1 was used to induce the migration of THP-1 cells, and the concentration of 500 ng/ml corresponds to data from the literature (Aiuti et al., 1997).

In this study, established pharmacological positive controls have not been included, since key experiments and the respective actions of active pharmacological compounds that substitute for a positive control have already been published by our group (Bräutigam et al., 2019; Stark et al., 2019; Bischoff-Kont et al., 2021; Krishnatas et al., 2021).

HUVECs were isolated according to Jaffe et al. (1973) or obtained from PELOBiotech (Planegg/Martinsried, Germany). For isolation of HUVECs, veins of human umbilical cords were exempted from residual blood clots with sterile phosphate-buffered saline (PBS) before a collagenase A (F. Hoffmann-La Roche, Basel, Switzerland) solution was filled into the vein and incubated for 45 min at 37°C to detach HUVECs. Subsequently, the umbilical vein was washed with medium 199 (PAN-Biotech, Aidenbach, Germany) containing 100 U/ml penicillin, 100 μg/ml streptomycin (PAN-Biotech, Aidenbach, Germany), and 10% fetal calf serum (FCS; Biochrom, Berlin, Germany), and the detached cells were centrifuged for 5 min at 300 g. The pellet was resuspended in an endothelial cell growth medium (ECGM) (EASY ECGM; PELOBiotech, Planegg/Martinsried, Germany) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin (PAN-Biotech, Aidenbach, Germany), 2.5 μg/ml amphotericin B (PAN-Biotech, Aidenbach, Germany), and 10% FCS (Biochrom, Berlin, Germany), and seeded onto a collagen G (Biochrom, Berlin, Germany)-coated cell culture flask and cultivated at 37°C in an atmosphere of 5% CO₂ and 95% air under constant humidity. HUVECs were split at a ratio of 1:3 and were used for experimental purposes exclusively as stated in passage 3.

The human monocytic cell line THP-1 and the human T cell line Jurkat were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). The cells were cultivated at 37°C in an atmosphere of 5% CO₂ and 95% air under constant humidity in the RPMI 1640 medium (PAN-Biotech) supplemented with 10% FCS (Biochrom, Berlin, Germany), and 100 U/ml penicillin, 100 μg/ml streptomycin (PAN-Biotech, Aidenbach, Germany), and 10% FCS (Biochrom, Berlin, Germany) were used in experiments up to passage 30.

HUVECs at 100% confluency were treated with the indicated concentrations of 6-shogaol or 6-gingerol for 30 min before the cells were activated with LPS (1 μg/ml), IL-1β (5 ng/ml), or TNF (10 ng/ml). After 24 h, THP-1 cells were fluorescence-labeled with CellTracker Green (Thermo Fisher Scientific, Schwerte, Germany), and 100,000 cells per well were allowed to adhere onto an endothelial cell monolayer. The amount of adherent THP-1 or Jurkat cells was measured at 485 nm (ex) and 535 nm (em) using a plate reader (SPECTRAFluor Plus, Tecan, Männedorf, Switzerland).

HUVECs were seeded into channel slides (µ-Slides I Luer, 0.8 mm; ibidi GmbH, Martinsried, Germany). Subsequently, the HUVECs in channel slides were connected to a pump system (ibidi GmbH, Martinsried, Germany), generating a one-directional flow (5 dyn/cm²) on the cells. After 24 h, when the cells had reached 100% confluency, HUVECs were treated with the indicated concentrations of 6-shogaol for 30 min before they were activated by LPS (24 h, 1 μg/ml). Fluorescence-labeled (CellTracker Green) THP-1 cells (8 × 10⁵ cells/ml) were allowed to adhere onto the 6-shogaol-treated and LPS-induced HUVEC monolayer in a one-directional flow of 0.5 dyn/cm². Adherent THP-1 cells on HUVECs were lysed using radioimmunoprecipitation assay (RIPA) buffer, and fluorescence was measured at 485 nm (ex) and 535 nm (em) using a plate reader (SPECTRAFluor Plus, Tecan, Männedorf, Switzerland).

Potential 6-shogaol-derived effects on leukocyte diapedesis through vascular endothelial cells were determined by performing a transmigration assay. For this, 100,000 HUVECs per well were seeded on collagen-coated Transwell membranes (Corning GmbH HQ, Glendale, AZ, United States, growth area 0.33 cm², 8 µm pore size, polycarbonate) in ECGM. After 24 h, when the cells reached a confluency of 100%, they were treated with the indicated concentrations of 6-shogaol for 30 min before they were activated with LPS (100 ng/ml). After 24 h, 2 × 10⁵ CellTracker Green-labeled THP-1 cells were allowed to transmigrate through the HUVEC monolayer in the direction of an SDF-1 gradient (500 ng/ml; Peprotech, Rocky Hill, NJ, United States) for 2 h. The upper side of the Transwell was exempted from non-transmigrated THP-1 cells using a cotton swab. Transmigrated THP-1 cells on the lower side of the Transwell were lysed in RIPA buffer and quantified by fluorescence measurement at 485 nm (ex) and 535 nm (em) using a plate reader (SPECTRAFluor Plus, Tecan, Männedorf, Switzerland).

For this assay, 1,500 HUVECs were seeded on collagen-G-coated 96-well plates in ECGM and cultivated for 24 h until they reached a confluency of 30%. Subsequently, the cells were treated with the indicated concentration of 6-shogaol for 72 h. Control cells were fixed using a methanol–ethanol (2:1) solution. After the end of the incubation period, the cells were fixed with methanol–ethanol and stained together with control cells using crystal violet (gentian violet; Sigma-Aldrich, St. Louis, MO, United States). After drying overnight, DNA-bound crystal violet was leached using 20% acetic acid. Absorbance was measured at 590 nm using a plate reader (SPECTRAFluor Plus, Tecan, Männedorf, Switzerland). Obtained data of control cells were subtracted from experimental values before they were further analyzed.

For the analysis of the impact of 6-shogaol on migration in the direction of a serum gradient, a Boyden chamber assay was performed. For this, 100,000 HUVECs were seeded on a collagen-coated Transwell membrane (Corning GmbH HQ, Glendale, AZ, United States; growth area 0.33 cm², 8 µm pore size, polycarbonate) in supplemented ECGM. The cells were allowed to adhere for 4 h to form a confluent layer (100%) before a serum gradient in medium 199 (0%–20%; PAN-Biotech, Aidenbach, Germany) was applied. At the same time, the cells were treated with indicated concentrations of 6-shogaol. HUVECs were allowed to migrate toward the serum gradient for 16 h before they were fixed (methanol:ethanol; 2:1) and stained using a crystal violet (Sigma-Aldrich, St. Louis, MO, United States) solution containing 20% methanol. Non-migrating cells on the upper side of the Transwell insert were thoroughly removed using a cotton swab. After drying overnight, cell-bound crystal violet was leached using 20% acetic acid and quantified by absorption measurement (590 nm) using a plate reader (SPECTRAFluor Plus, Tecan, Männedorf, Switzerland).

Further insights into the migratory behavior of cells can be provided by the performance of a 2D chemotactic migration assay. Therefore, 18,000 HUVECs were seeded on chemotaxis slides (ibidi GmbH, Martinsried, Germany) in ECGM and were allowed to adhere for 4 h in order to obtain confluency of about 10% before they were treated with indicated concentrations of 6-shogaol. The cells were allowed to migrate toward a serum gradient (FCS, 20% in medium 199) for 20 h. Live cell imaging in an atmosphere of 5% CO₂ and 95% air at 37°C, capturing an image every 10 min using a microscope (DM IL LED, Leica Microsystems, Wetzlar, Germany) allowed single-cell tracking. Image quantification of 30 cells per donor and condition using a manual-tracking tool (ImageJ) indicated a detailed 6-shogaol-derived impact on the migration parameters accumulated distance, Euclidean distance, velocity, forward migration index Y (Y:FMI), and directness.

Endothelial cell spheroids from HUVECs were generated in a supplemented ECGM medium containing 0.24% methylcellulose by using the hanging drop method. Subsequently, the spheroids were embedded into a rat tail collagen I gel. HUVEC sprouting was induced by treating with vascular endothelial growth factor (VEGF; PeproTech, Rocky Hill, NJ, United States) for 20 h in an endothelial cell basal medium (ECBM; PELOBiotech, Planegg/Martinsried, Germany). For the determination of 6-shogaol-derived effects on endothelial cell sprouting, HUVEC spheroids were treated with the indicated concentrations of the compound 30 min prior to VEGF induction. After the incubation period, the spheroids were fixed with 4% Roti Histofix (Carl Roth, Karlsruhe, Germany) before microscopic images were taken (DM IL LED, Leica Microsystems, Wetzlar, Germany). Image analysis using ImageJ (software version 1.49k) allowed the quantification of endothelial cell sprouts.

Organ (mouse aortae) removal was performed according to the German Animal Welfare Act (§ 4 Tierschutzgesetz) and approval number V54-19c20/21I-FR/Biologicum, Tierhaus Campus Riedberg (Regierungspräsidium Darmstadt, Germany). Animals (C57BL/6 N mice) were housed under a 12 h light/dark cycle with access to food and water ad libitum.

To analyze the effect of 6-shogaol on angiogenic processes an ex vivo aortic ring assay was performed (Baker et al., 2011). For this, 3–5 week old C57BL/6 mice (male/female) were sacrificed by carbon dioxide (CO₂) exposure. Subsequently, aortae were dissected, and the adjacent soft tissue was removed before the blood vessels were cut into rings with an average size of 0.5–1 mm. Aortic rings were serum-starved overnight in OPTI-MEM I (Thermo Fisher Scientific, Schwerte, Germany) containing 100 U/ml penicillin and 100 μg/ml streptomycin (PAN-Biotech). The next day aortic rings were embedded into 50 µl rat tail collagen I gel (Corning GmbH HQ; 1.5 mg/ml). After gel solidification, aortic rings were treated with 30 ng/ml murine VEGF (Peprotech, Rocky Hill, NJ, United States) in OPTI-MEM (Thermo Fisher Scientific, Schwerte, Germany), supplemented with 2.5% FCS (Biochrom, Berlin, Germany), 100 U/ml penicillin, and 100 μg/ml streptomycin (PAN-Biotech, Aidenbach, Germany) to induce endothelial sprouting. When initial sprouts were visible, aortic rings were treated with 6-shogaol (30 µM) or vehicle, i.e., 0.03% dimethyl sulfoxide (DMSO), and 30 ng/ml VEGF for additional 3 days. For the analysis of potential 6-shogaol-derived vascular-disruptive effects, endothelial sprouts were allowed to form for 6–9 days, before the aortic rings were treated with 6-shogaol (30 µM) or vehicle (0.03% DMSO) and 30 ng/ml VEGF for 24 or 48 h.

After the respective incubation periods, aortic rings and sprouts were fixed using 4% Roti Histofix (Carl Roth, Karlsruhe, Germany) and permeabilized using Triton-X100 (Carl Roth, Karlsruhe, Germany) two times for 15 min at room temperature. The sprouts were stained using BS-I lectin (FITC, 0.1 mg/ml; lectin from Bandeiraea simplicifolia (Griffonia simplicifolia); #2895; Sigma-Aldrich; St. Louis, MO, United States) and actin α-smooth muscle (CY3; mouse, anti-mouse, 1:1,000; #C6198; Sigma-Aldrich; St. Louis, MO, United States) at 4°C overnight. Sprouts from murine aortic rings were analyzed using laser confocal scanning microscopy (Zeiss LSM 780; Carl Zeiss, Oberkochen, Germany) and quantified by ImageJ (software version 1.49k; US National Institutes of Health, Bethesda, MD, United States).

Confluent HUVECs (100% confluency) were treated with indicated concentrations of 6-shogaol. 30 min later, the cells were activated with LPS (1 μg/ml) for 4 h (E-selectin) or 24 h (intercellular adhesion molecule, ICAM-1; vascular cell adhesion molecule, VCAM-1), before they were stained for ICAM-1 (fluorescein isothiocyanate, FITC; mouse, anti-human CD54, 1:33, MCA1615F; Bio-Rad, Hercules, CA, United States), VCAM-1 (phycoerythrin, PE; mouse, anti-human CD106, 1:20, 555647; BD Biosciences, San Jose, CA, United States), or E-selectin (PE, mouse, anti-human CD62E, 1:10, 551145; BD Biosciences, San Jose, CA, United States). Levels of the respective surface protein were determined using flow cytometry (FACSVerse; BD Biosciences, San Jose, CA, United States).

Confluent HUVECs (100% confluency) were treated with the indicated concentrations of 6-shogaol for 30 min before they were activated with LPS (1 μg/ml) for 4 (E-selectin) or 24 h (ICAM-1, VCAM-1). Subsequently, the cells were lysed using RIPA buffer containing protease and phosphatase inhibitors. The total protein in each sample was determined using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Schwerte, Germany), and a pyronin-based sample buffer containing sodium dodecyl sulfate (SDS) was added before they were incubated at 95°C for 5 min for denaturation. Twenty-five µg of each sample was subjected to discontinuous SDS-polyacrylamide gel electrophoresis (PAGE) for protein separation, before they were transferred onto a polyvinylidene fluoride (PVDF; Bio-Rad Laboratories, Munich, Germany) membrane by blotting for 1 h at 100 V or 16 h at 30 V (VEGFR2). For blocking of unspecific binding sites, the membranes were incubated for 2 h in 5% Blotto (Carl Roth, Karlsruhe, Germany) or 5% bovine serum albumin (BSA; MilliporeSigma, Burlington, MA, United States) containing 0.1% Tween-20 (Sigma-Aldrich; St. Louis, MO, United States). Subsequently, the membranes were incubated with respective antibodies for detection of the protein of interest: anti-ICAM-1 (rabbit, anti-human, 1:1,000 in 5% Blotto + Tween-20, 4°C overnight, #4915; Cell Signaling/New England Biolabs, Frankfurt am Main, Germany), anti-VCAM-1 (mouse, anti-human, 1:1,000 in 5% Blotto + Tween-20, 4°C overnight, sc-13160; Santa Cruz Biotechnology, Dallas, TX, United States), anti-E-selectin (mouse, anti-human, 1:2,000 in 5% Blotto + Tween-20, 4°C overnight, sc-137054; Santa Cruz Biotechnology), anti-toll-like receptor (TLR) 4 (mouse, anti-human, 1:400 in 5% Blotto + Tween-20, 4°C overnight, sc-293072 Santa Cruz Biotechnology, Dallas, TX, United States), anti-VEGFR2 (rabbit, anti-human, 1:2,000 in 5% BSA + Tween-20, 4°C overnight, #9698; Cell Signaling/New England Biolabs, Frankfurt am Main, Germany), and anti-HO-1 (P249) (rabbit, anti-human, 1:1,000 in 5% BSA + Tween-20, 4°C overnight, #5061; Cell Signaling/New England Biolabs, Frankfurt am Main, Germany). Anti-β-actin-peroxidase (mouse, anti-human, 1:50,000 in 1% BSA + Tween-20, 1 h room temperature, A3854; Sigma-Aldrich, St. Louis, MO, United States) was used to detect β-actin as the loading control. Secondary antibodies were applied to membranes being incubated with non-conjugated primary antibodies: anti-rabbit (goat, 1:2,000 in 5% Blotto + Tween-20, 2 h room temperature, #7074; Cell Signaling/New England Biolabs, Frankfurt am Main, Germany) and anti-mouse, HRP-linked antibody (horse, 1:2,000 in 5% Blotto + Tween-20, 2 h room temperature, #7076; Cell Signaling/New England Biolabs, Frankfurt am Main, Germany). Protein detection was achieved by chemiluminescence measurement, and quantification of specific protein levels in each sample was performed by densitometric analysis using ImageJ (software version 1.49k).

Confluent HUVECs (100% confluency) on 8-well µ-slides (ibidi GmbH, Martinsried, Germany) were treated with indicated concentrations of 6-shogaol for 24 h before they were activated with LPS (1 μg/ml) for indicated time points. Subsequently, the cells were fixed with 4% Roti Histofix (Carl Roth, Karlsruhe, Germany) before they were permeabilized using 0.2% Triton-X100 (Carl Roth). After blocking unspecific binding sites using 0.2% BSA (MilliporeSigma, Burlington, MA, United States), the cells were incubated in a 0.2% BSA solution containing the primary antibody anti-NFκB-p65 (rabbit, anti-human, 1:400, sc-372; Santa Cruz Biotechnology, Dallas, TX, United States). After 1 h the primary antibody was removed, and the secondary antibody (goat, anti-rabbit, Alexa Fluor 488-conjugated, 1:400, A11008; Thermo Fisher Scientific, Schwerte, Germany) was incubated for 45 min protected from light. Immunofluorescence staining of NFκB-p65 was determined using a Leica DMI6000B fluorescence microscope (Leica Microsystems, Wetzlar, Germany). The use of ImageJ (software version 1.49k; US National Institutes of Health, Bethesda, MD, United States) allowed the quantification of NFκB-p65 translocation.

Confluent HUVECs (100% confluency) were treated with indicated concentrations of 6-shogaol for 30 min, before they were activated with LPS (1 μg/ml) for 2 h (E-selectin) or 10 h (ICAM-1, VCAM-1). RNA was isolated using the RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions, and 1 µg was transcribed into cDNA using the SuperScript reverse transcriptase (Life Technologies, Darmstadt, Germany). The 2^−ΔΔCT method was used for quantitative real-time PCR data analysis. GAPDH served as the housekeeping gene, and the following primers were used: E-selectin (forward: 5′-AGA-TGA-GGA-CTG-CGT-GGA-GA-3′; reverse: 5′-GTG-GCC-ACT-GCA-GGA-TGT-AT-3′), VCAM-1 (forward: 5′-CCA-CAG-TAA-GGC-AGG-CTG-TAA-3′; reverse: 5′-GCT-GGA-ACA-GGT-CAT-GGT-CA-3′), ICAM-1 (forward: 5′-CTG-CTC-GGG-GCT-CTG-TTC-3′; reverse: 5′-AAC-AAC-TTG-GGC-TGG-TCA-CA-3′), VEGFR2 (forward: 5′-GTG-ACC-AAC-ATG-GAG-TCG-TGT-3′; reverse: 5′-AGC-TGA-TCA-TGT-AGC-TGG-GAA-3′), and GAPDH (forward: 5′-CCA-CAT-CGC-TCA-GAC-ACC-AT-3′; reverse: 5′-TGA-AGG-GGT-CAT-TGA-TGG-CAA-3′).

For this assay, 1 × 10⁶ HUVECs (PeloBiotech, Planegg/Martinsried, Germany) per aluminum cuvette were transiently transfected with a Renilla luciferase control plasmid pGL4.74[hRluc/TK] and a firefly luciferase reporter plasmid pGL4.32[luc2P/NFκB-RE/Hygro] (Promega, Heidelberg, Germany) at a ratio of 5:10 by electroporation (Amaxa nucleofector 2b device; Lonza, Basel, Switzerland). Twenty-four h after transfection, when HUVECs reached a density of 100% (confluency), the cells were treated with the indicated concentrations of 6-shogaol or 6-gingerol for 6 h before they were activated by LPS for 4 h (NFκB). Afterward, HUVECs were exposed to passive lysis according to the manufacturer’s instructions, and NFκB promotor activity was determined using the Dual-Luciferase Reporter Assay System (Promega, Heidelberg, Germany) using a luminometer (SPECTRAFluor Plus, Tecan, Männedorf, Switzerland).

All experiments were performed with at least three different HUVEC or aortae donors (n). The actual number of n is stated in the respective figure legend. Statistical analyses were performed using GraphPad Prism version 5.0 (San Diego, CA, United States) by applying one-way ANOVA and Tukey’s post hoc test. For the ex vivo assay, an unpaired Student’s t-test followed by Welch’s correction was used. Data are expressed as mean ± standard deviation (SD) and considered as statistically significant when p ≤ 0.05.

Studying the potential of 6-shogaol to interfere with this process in endothelial cells (ECs), we compared the impact of the compound on the adhesion of the monocytic THP-1 cell line onto compound-treated and TNF-, IL-1β-, or LPS-activated HUVECs under static conditions.

In our study, only HUVECs were treated with 6-shogaol or 6-gingerol (Supplementary Figure S1). Analyzing potential cytotoxic effects, we found that up to 30 µM of 6-shogaol incubated over a period of 24, 48, and 72 h did not impair endothelial cell viability nor membrane integrity (Supplementary Figures S2A,B). Therefore, for experimental purposes, concentrations of no more than 30 µM were used. For the exclusion of potential cytotoxic effects of 6-gingerol, HUVECs were treated with increasing concentrations for 24 and 48 h. Up to 100 µM did not impair the cell viability (Supplementary Figure S2C).

6-Shogaol at a concentration of 30 µM significantly reduced the adhesion of THP-1 cells onto a TNF-, IL-1β-, or LPS-triggered HUVEC monolayer under static conditions (Figure 1A). When TNF was used as a pro-inflammatory stimulus, a concentration of 30 µM of 6-shogaol was able to almost fully block the adhesion. In HUVECs that were treated by IL-1β or LPS, 6-shogaol was already active at a concentration of 10 µM. To determine if 6-shogaol was able to reduce the adhesion of leukocytes onto an LPS-induced HUVEC monolayer under the flow conditions, a pump system was used to mimic the blood flow. 6-shogaol was able to reduce the adhesion of THP-1 cells onto LPS-activated HUVECs significantly at 10 and 30 µM. Of note, treatment of HUVECs with 30 µM 6-shogaol completely blocked the adhesion of THP-1 cells under flow conditions (Figure 1B). In addition to 6-shogaol, 6-gingerol has been described as one of the main bioactive ingredients of ginger rhizomes and represents the hydrated precursor of 6-shogaol missing the Michael acceptor moiety. Interestingly, the application of up to 100 µM of 6-gingerol did not reduce THP-1 adhesion onto LPS-induced HUVECs (Figure 1A).

Furthermore, the adhesion of the leukemia cell line Jurkat with T-lymphoblast-like phenotype was significantly reduced in LPS-activated HUVECs when treated with 30 µM 6-shogaol (Supplementary Figure S3). Moreover, and of high importance, 6-shogaol reduced the transmigration of THP-1 cells through LPS-triggered HUVECs in the direction of an SDF-1-gradient. This effect was already significant at 10 µM and completely blocked by 30 µM 6-shogaol (Figure 1C).

As 6-shogaol reduced the interaction of THP-1 and Jurkat cells and activated HUVECs, the levels of endothelial cell adhesion molecules (CAMs) on the endothelial surface, their total amount, and mRNA expression were determined. 6-Shogaol significantly reduced TNF-induced ICAM-1, VCAM-1, and E-selectin levels on the endothelial cell surface (Supplementary Figure S4A). The determination of 6-shogaol-derived effects revealed that also the total protein levels of CAMs were reduced, and the most prominent effect was observed at 30 µM (Supplementary Figure S4B). The analysis of mRNA levels revealed that increasing concentrations of 6-shogaol reduced the gene expression of ICAM-1, VCAM-1, and E-selectin in a concentration-dependent manner (Supplementary Figure S4C). Similar effects were observed after 6-shogaol treatment, when HUVECs were activated with the inflammatory cytokine IL-1β. Here, the most prominent inhibitory effects were also detectable at 30 µM (Supplementary Figures S5A–C). Interestingly, when LPS was used for the inflammatory activation of HUVECs, 6-shogaol significantly reduced surface levels of ICAM-1, VCAM-1, and E-selectin already at 10 µM. Of note, the application of 30 µM was able to completely block the surface levels of CAMs (Figure 2A). In addition, the effects on total protein and mRNA levels were much stronger for the activation with LPS compared to the application of the cytokines TNF or IL-1β. Here, 6-shogaol significantly reduced CAM levels at 10 µM and blocked their expression at 30 µM (Figures 2B,C).

The effect of 6-shogaol on the activity of NFκB was analyzed in LPS-induced HUVECs. Moreover, to compare the effect of 6-gingerol on the NFκB-signaling cascade, HUVECs were treated with the indicated concentrations of 6-gingerol before they were analyzed for the LPS-induced promotor activity of the transcription factor. Interestingly, the promotor activity of NFκB was strongly downregulated by 6-shogaol in a concentration-dependent manner and completely blocked by a compound concentration of 30 μM, while 6-gingerol did not impair the promotor activity of the transcription factor (Figures 3A,B). Moreover, LPS-induced inflammatory target genes of NFκB were downregulated by 6-shogaol. The mRNA levels of the pro-inflammatory cytokines IL-6 and IL-8 were significantly reduced in a concentration-dependent manner (Supplementary Figure S6A). The mRNA expression of the LPS-triggered chemokine C-X-C motif chemokine ligand 12 (CXCL12) was significantly reduced at 30 µM 6-shogaol (Supplementary Figure S6B). Interestingly, mRNA levels of the anti-inflammatory cytokine IL-10 remained unimpaired (Supplementary Figure S6C).

Mitogen-activated protein kinases (MAPKs) were implicated in the NFκB-signaling cascade, and Western blot analysis revealed that the LPS-induced c-Jun N-terminal kinase (JNK) phosphorylation was markedly reduced upon 6-shogaol (30 µM) treatment, while the LPS-triggered p38 activation was not impaired (Supplementary Figure S6D).

As the 6-shogaol-derived inhibition of LPS-induced NFκB promotor activity might be attributed to an impairment of nuclear translocation, the amount of the NFκB subunit p65 was analyzed in HUVEC nuclei after the treatment with 6-shogaol (30 µM) by immunofluorescence staining (Figure 3C). Interestingly, only after 1 h of LPS induction, 6-shogaol reduced the nuclear translocation of p65 by 35%. Analyzing the effect of 6-shogaol (30 µM) on the toll-like receptor 4 upstream the LPS-activated NFκB-signaling cascade, we found that the protein levels remained unimpaired (Figure 3D).

HO-1 protein levels were markedly increased in LPS-stimulated HUVECs upon 6-shogaol (30 µM) treatment (Figure 3E).

The application of 6-shogaol resulted in a concentration-dependent reduction of HUVEC proliferation with an IC₅₀ of 8 µM (Figure 4A). Moreover, the directed HUVEC migration toward a serum gradient was significantly reduced upon 6-shogaol treatment at 10 and 30 µM (Figure 4B). Next, we aimed at gaining detailed information about the impact of the ginger constituent on endothelial cell migration in a 2D chemotaxis assay via live-cell imaging. Here, HUVECs were allowed to migrate in the direction of a serum gradient over a period of 20 h. By single-cell tracking, detailed data were obtained about the cell motility upon 6-shogaol (30 µM) treatment. Indeed, the main characteristics of endothelial cell migration were impaired as indicated by significantly reduced directness and velocity. Moreover, 6-shogaol attenuated Euclidean and accumulated distances. The most prominent effect was exposed on the forward migration index (FMI:Y) (Figure 4C).

To determine the effect of 6-shogaol on endothelial cell sprouting, spheroids were formed from HUVECs, and the cells were allowed to sprout in a 3D rat tail collagen I gel after VEGF induction. Cells in spheroids were treated with the indicated concentrations of 6-shogaol for 30 min, before VEGF was applied to trigger sprout formation. As depicted in Figure 5A, VEGF successfully induced sprout formation as demonstrated by a strong increase in sprout number and sprout length. The administration of 3 and 10 µM 6-shogaol only slightly reduced the formation of new sprouts. However, 30 µM of 6-shogaol completely blocked endothelial cell sprouting as indicated by a strongly inhibited number of sprouts and reduced sprout length. Of note, the number and length of VEGF-activated sprouts were much lower in 6-shogaol (30 µM)-treated HUVEC spheroids than in the untreated control implicating a compound-induced loss of endothelial cell functions necessary for sprouting processes.

As the VEGF-induced endothelial cell sprouting from HUVEC spheroids was completely blocked by 6-shogaol (30 µM), the effect of the ginger constituent on the total VEGFR2 protein was analyzed. 6-Shogaol (30 µM) markedly reduced the levels of the receptor after 6 and 10 h of treatment, and this impact ceased after 16 h of incubation (Figure 5B). qPCR analysis revealed that the mRNA expression of VEGFR2 was significantly reduced after 3 and 6 h. This indicates that the 6-shogaol-derived downregulation of VEGFR2 might, at least in part, be ascribed to attenuated mRNA levels (Figure 5C).

To obtain detailed information about the 6-shogaol-derived antiangiogenic potential in a physiologically more relevant model, a mouse aortic ring assay was performed. As depicted in Figure 5D, angiogenic sprouts were clearly formed in the vehicle-treated group. FITC-BS-I lectin (green) staining visualizes endothelial sprouting and CY3 (red) indicates actin α-smooth muscle-positive cells. The administration of 6-shogaol resulted in a strong and significant inhibition of VEGF-induced angiogenic sprout formation as indicated by a significantly attenuated number of sprouts and total sprout length per aortic ring.

Interestingly, 6-shogaol was also able to interfere with already existing neo-vessels formed from VEGF-induced murine aortic rings (Figure 6). The sprout formation in the VEGF control was significantly increasing after 48 h compared to 0 h (Figures 6A,B). Already after 24 h of 6-shogaol treatment (30 µM), the compound disrupted existing sprouts, and this effect was significant compared to the control group treated with VEGF alone and, moreover, to the starting point of administration (0 h) as indicated by the reduction of single sprout length (Figure 6A). Of note, after 48 h angiogenic sprouts were restituted (Figures 6A,C).