2016 Volume 39 Issue 6 Pages 1029-1034
Due to its powerful ability to deplete cholesterol from the plasma membrane of cells, methyl-β-cyclodextrin (MβCD) has been widely used as a putative research tool in cell biology. Recently, recruiting MβCD as an effective drug (e.g., antitumor drugs) has been developed. However, it remains unclear whether MβCD, when it enters the blood circulation as a drug, influences the functions of the endothelium, e.g., the adhesion of leukocytes to the endothelium. In this study, we found that MβCD can impair the adhesion of monocytes to the monolayer of endothelial cells by lowering the cell-surface adhesive force and expression of adhesion molecules and caveolae-related molecules on/in endothelial cells, and reorganizing the actin cytoskeleton of endothelial cells. The data imply that MβCD, when recruited as a drug, potentially helps to inhibit inflammation or initiation/progression of atherosclerosis since its important early step is the adhesion of circulating leukocytes (e.g., monocytes) to the endothelium.
Beta-cyclodextrin (β-CD) is a cyclic oligosaccharide consisting of seven α-(1,4)-linked glucopyranose subunits. Due to its relatively low water solubility (approximately 2% weight by weight),1) various derivatives of β-CD have been developed to significantly improve the water solubility, among which methyl-β-cyclodextrin (MβCD) is the one studied most intensively. MβCD is well known as a putative powerful cholesterol-depleting agent and commonly used to deplete plasma membrane cholesterol from cultured mammalian cells.2–4) MβCD-cholesterol complexes also can efficiently replenish cells with cholesterol as cholesterol donors.3,4)
Moreover, due to the structure of a lipophilic internal cavity surrounded by hydrophilic outer surface, MβCD was applied as a drug delivery system.5) Recently, it was found that MβCD per se potentially has antitumor effects and has the possibility of being an effective antitumor drug.6–11) However, it remains uncertain whether MβCD, as an anticancer drug, influences the functions of the endothelium, e.g. the adhesion of circulating leukocytes (e.g. monocytes) onto the endothelium which is an important step during inflammation or initiation/progression of atherosclerosis.
In this study, the effect of MβCD on the adhesion of monocytes onto the monolayer of endothelial cells was first evaluated. Then, the effects of MβCD on cell-surface adhesive force properties, expression/distribution of adhesion molecules and caveolae-related molecules, and actin cytoskeleton of endothelial cells were investigated to elucidate the potential underlying mechanisms.
Human umbilical vein endothelial cells (HUVECs) were purchased from Xiangya Central Experiment Laboratory (Hunan, China) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, U.S.A.) supplemented with 10% (w/v) fetal calf serum (Hyclone, South Logan, UT, U.S.A), 100 U/mL penicillin, and 100 µg/mL streptomycin. For all experiments, cell cultures had been passaged approximately 5 times.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) AssayMTT assay was performed as reported previously.12) Approximately 8×103 HUVECs were plated in each well of a 96-well plate and cultured at 37°C in a 5% CO2 incubator for 24 h. After washing with phosphate buffered saline (PBS), cells in different wells were treated with different concentrations (0, 5, 10, 20 mM) of MβCD (Sigma, U.S.A.) for 30 min at 37°C. After PBS washes, 100 µL of fresh medium and 20 µL 5 mg/mL MTT were added to each well, and the cells were incubated for 4 h. After reaction termination and solution removal, 100 µL of dimethyl sulfoxide (DMSO) was added to each well, and the plate was shaken gently at 37°C for 10 min. The absorbance (optical density) was measured at 578 nm with a microplate reader (Rayto).
Monocyte Adhesion AssayCelltracker C34552 (Invitrogen, U.S.A.) was utilized to stain THP-1 cells for ca. 30 min at 37°C according to the manufacturer’s instruction. The cells were washed twice with serum-free medium to remove excess dyes. Then the cells were added onto the confluent monolayer of HUVECs pretreated with or without 1, 5, and 10 mM MβCD for 30 min at 37°C and incubated for 2 h at 37°C. After washing twice with PBS to remove the unattached cells, the samples were observed under a confocal microscope. The excitation and emission wavelengths for Celltracker C34552 were 577 and 602 nm, respectively. The number of attached fluorescently-stained THP-1 cells in each field was counted.
Adhesive Force Mapping by Atomic Force Microscopy (AFM)The living HUVECs treated with or without 10 mM MβCD for 30 min at 37°C were immediately detected by Agilent series 5500 AFM in contact mode. The adhesive force mapping of an area of 30×30 µm on the cell surface was performed. The length and width of the AFM cantilevers were 200 and 28 µm, respectively, with a resonant frequency of 77 kHz and a spring constant of ca. 0.04 N/m.
Confocal Microscopy and Flow CytometryFor fluorescence imaging, an LSM710 confocal microscope (Carl Zeiss, Oberkochen, Germany) equipped with a Zeiss inverted microscope and a Zeiss Plan-Neofluar objective (40×/0.75) was recruited. Flow cytometric acquisition and analysis were performed in a FACSCalibur flow cytometer (BD Biosciences, U.S.A.).
Fluorescence Staining of Adhesion Molecules, Caveolin-1, and Phosphatidylinositol 4,5-Bisphosphate (PIP2)The fluorescence staining of each molecule was performed according to the manufactory provided protocol. Briefly, the cells pre-treated with or without MβCD for 30 min at 37°C were fixed with 4% paraformaldehyde for 20 min at room temperature, washed with PBS (for adhesion molecules and caveolin-1) or Tris-buffered saline (TBS); for PIP2) two times, and then subjected to fluorescence staining.
For the surface staining of intercellular adhesion molecule 1 (ICAM-1), the fixed cells were incubated with phycoerythrin (PE)-conjugated anti-human CD54 (eBioscicence, CA, U.S.A.) at a final concentration of 5 µg/mL in PBS for 1 h at 37°C. After washing with PBS three times, the samples were subjected to flow cytometry.
For caveolin-1 staining, the fixed cells were incubated with 5 µg/mL rabbit anti-caveolin-1 (Abcam, London, U.K.) in PBS plus 0.05% saponin for 1 h at 37°C and then with Pacific blue-conjugated goat anti-rabbit immunoglobulin G (IgG) (Life Technologies, U.S.A.) in PBS (1 : 1000) for 30 min at 37°C. After washing with PBS three times, the samples were subjected to confocal microscopy and flow cytometry.
For PIP2, the intracellular staining was performed as reported previously.13) The fixed cells were permeabilized with 0.5% saponin for 15 min at room temperature, washed three times with TBS, and blocked with 1% bovine serum albumin (BSA) in TBS over night at 4°C. Then, the cells were stained with biotinylated mouse anti-PIP2 IgM (Echelon Biosciences, UT, U.S.A.) at a final concentration of 10 µg/mL in TBS for 1 h at 37°C and subsequently with streptavidin-conjugated Alexa Fluor 555 (Life Technologies, U.S.A.) in TBS for 30 min at 37°C. After washing thoroughly with distilled water, the cells were subject to confocal microscopy and flow cytometry.
Visualization and Quantification of Actin CytoskeletonHUVEC Cells were plated in petri dishes and cultured in the incubator (37°C, 5% CO2) for 24 h to reach a 70% confluence. After treated with or without 10 mM MβCD for 30 min at 37°C, the cells were fixed by 4% paraformaldehyde for 20 min, washed three times with PBS, and stained by anti-paxillin monoclonal antibody (mAb) (overnight at 4°C; Abcam) plus Alexa fluor 488 goat anti-mouse IgG (for 2 h at 37°C) and tetramethylrhodamine (TRITC)-Phalloidin (for 1 h at room temperature; Sigma) as usual. After washing twice with PBS, the cells were observed under the confocal microscope. The excitation/emission wavelengths for Alexa fluor 488 and TRITC were 488 nm/(493–630 nm) and 561 nm/(570–670 nm), respectively. The length of each fluorescently labeled actin filament was measured using the Zeiss LSM 710 Zen software.
Cell Spreading AssaySpread area-based spreading assay was performed as reported previously.14,15) Briefly, HUVECs at 70% confluence were treated with or without 10 mM MβCD for 30 min at 37°C. The cells were then fixed by 4% paraformaldehyde, rinsed with PBS, and imaged in PBS by the confocal microscope. The spread area of each cell was extracted by planimetry via the Zeiss LSM 710 Zen software.
Statistical AnalysisAll graphs were made using GraphPad Prism version 5.0 (GraphPad Software, La Jolla, CA, U.S.A.) on which all data were expressed as the mean±standard deviation (S.D.) from at least three independent experiments. Statistical analysis was performed using Student’s t-test. A difference was regarded as significant when p<0.05 (* p<0.05; ** p<0.01; *** p<0.001).
The effects of MβCD on the membrane alternation, cytoskeletal reorganization, proliferation, and apoptosis of endothelial cells have been intensively studied.16–18) However, it remains unclear whether and how MβCD affects the adhesion of leukocytes (e.g. monocytes) onto the endothelium which is the first step of leukocyte trans-endothelial migration.
Firstly, we studied the effects of MβCD at different concentrations on the viability of HUVECs using MTT assay (Fig. 1). The data shows that the 20 mM MβCD treatment for 30 min induced significant death of HUVECs whereas ≤10 mM MβCD treatments for 30 min caused no statistically significant, although slight, decrease in cell viability. Therefore, in the following experiments we used ≤10 mM MβCD to treat HUVECs for 30 min.
To test whether MβCD influence the adhesion of monocytes onto endothelial cells, THP-1 cells (a putative monocyte cell line) were fluorescently stained with Celltracker, co-cultured with the monolayer of HUVEC cells (a widely used endothelial cell line) pretreated with or without 1, 5, and 10 mM MβCD, respectively and imaged by confocal microscopy. The confocal fluorescence images clearly show that compared with untreated HUVECs there were less THP-1 cells on the monolayer of the HUVECs treated by 5 or 10 mM MβCD (Fig. 2A). It was further confirmed by the statistical analysis (Fig. 2B), implying that MβCD significantly inhibited the adhesion of THP-1 cells onto HUVECs.
Celltracker C34552 was utilized to stain THP-1 cells. (A) Fluorescently stained THP-1 cells on the monolayer of HUVECs treated with 0, 1, 5, and 10 mM MβCD for 30 min, respectively. (B) Average numbers of THP-1 cells adhered on the monolayer of HUVECs treated with or without MβCD in a field with the same size.
Since the biomechanical properties of cell surfaces may be involved in cell-cell adhesion, the cell-surface adhesive force of HUVECs treated with or without 10 mM MβCD was then detected by the force measurement function of AFM. The adhesive force mapping on a cell surface region of 30×30 μm shows that MβCD can impair the cell-surface adhesive force of HUVECs (Fig. 3).
(A) Representative adhesive force mapping on an untreated cell detected by AFM. (B) Representative adhesive force mapping on a MβCD (10 mM)-treated cell detected by AFM. Scan size: 30 µm×30 µm.
It is well known that adhesion molecules on cell surfaces are the major determinants of the adhesion of leukocytes onto the endothelium. Therefore, we then investigated the effects of MβCD on the expression of ICAM-1 on HUVECs by using flow cytometry (Fig. 4). The data shows that the mean fluorescence intensity (MFI) of ICAM-1 on HUVECs significantly decreased after treated with 5 and 10 mM MβCD. The data implies that MβCD can inhibit the expression of adhesion molecules on HUVECs in a dose-dependent manner.
Left: representative flow cytometric data showing the effects of MβCD on the mean fluorescence intensity (MFI) of fluorescently stained ICAM-1 on HUVECs. Right: quantification and statistical analyses of MβCD-induced changes in MFI of ICAM-1.
Caveolae, a special type of cholesterol-enriched lipid raft, are highly abundant in endothelial cells and play multiple roles in cell adhesion and signal transduction.19) Another important molecule PIP2 which plays a string-puller role in membrane-cytoskeleton coupling20,21) is also enriched in caveolae.22,23) Therefore, we tried to evaluate the effects of MβCD on caveolae and PIP2. The data from both confocal microscopy and flow cytometry indicate that MβCD also inhibited the expressions of caveolin-1 (Fig. 5), a structural protein of caveolae, and PIP2 (Fig. 6).
Finally, we sought to investigate the effects of MβCD on actin cytoskeleton which is associated with plasma membrane generally via caveolae/PIP2. The data show that MβCD induced significant decrease in the average length of individual actin filaments causing the decrease in the average spreading area of individual HUVECs (Fig. 7). Therefore, MβCD also caused cytoskeletal reorganization/shortening in HUVECs probably due to the MβCD-induced weakening of the caveolae/PIP2 which mediates the membrane-cytoskeleton coupling.
(A) The representative (left) and statistical analyses (right) of flow cytometric data show that the MFI of caveolin-1 in MβCD-treated cells was significantly lower than that on untreated cells. (B) Confocal microscopy shows that compared with untreated cells (upper panel) there were fewer caveolin-1 molecules in the treated cells by 10 mM MβCD (lower panel). From left to right: DIC image, fluorescence image, and merged image, respectively.
Confocal microscopy shows that compared with untreated cells (upper panel) there were fewer PIP2 molecules in the treated cells by 10 mM MβCD (middle panel). From left to right: DIC image, fluorescence image, and merged image, respectively. The representative (left) and statistical analyses (right) of flow cytometric data (bottom panel) show that the MFI of PIP2 on MβCD-treated cells was significantly lower than that on untreated cells.
Left panel: a representative fluorescence confocal image showing actin filaments and focal adhesions at both ends of filaments by fluorescently staining F-actin (red) and paxillin (a main focal adhesion molecule; green), respectively. Middle panel: the average length of individual actin filaments in HUVECs treated with or without MβCD. Right panel: the average spreading area of individual HUVECs treated with or without 10 mM MβCD.
Taken together, in a concentration-dependent manner MβCD can inhibit the adhesion of monocytes onto endothelial cells by down-regulating the cell-surface adhesive force property and the expression of adhesion molecules on endothelial cells which is probably correlated with caveolae (or lipid rafts) and cytoskeletal reorganization although it still remains controversial whether adhesion molecules (e.g. ICAM-1) are present in caveolae.24–28) It implies that MβCD, when recruited as an anticancer drug, will impair the adhesion of circulating leukocytes (e.g. monocytes) onto the endothelium and therefore potentially help to inhibit inflammation or initiation/progression of atherosclerosis.
This study was supported by the National Natural Science Foundation of China (81560083) and the Natural Science Foundation of Jiangxi Province (20151BAB205005).
The authors declare no conflict of interest.
