Accepted Manuscript
Barrier protective effect of asiatic acid in TNF-α-induced activation of human aortic endothelial cells Lai Yen Fong , Chin Theng Ng , Zhi Li Cheok , Mohamad Aris Mohd Moklas , Muhammad Nazrul Hakim , Zuraini Ahmad PII: DOI: Reference:
S0944-7113(15)00384-0 10.1016/j.phymed.2015.11.019 PHYMED 51943
To appear in:
Phytomedicine
Received date: Revised date: Accepted date:
14 October 2015 17 November 2015 26 November 2015
Please cite this article as: Lai Yen Fong , Chin Theng Ng , Zhi Li Cheok , Mohamad Aris Mohd Moklas , Muhammad Nazrul Hakim , Zuraini Ahmad , Barrier protective effect of asiatic acid in TNF-α-induced activation of human aortic endothelial cells, Phytomedicine (2016), doi: 10.1016/j.phymed.2015.11.019
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Barrier protective effect of asiatic acid in TNF-α-induced activation of human aortic endothelial cells
Muhammad Nazrul Hakima and Zuraini Ahmada,*
a
CR IP T
Lai Yen Fonga, Chin Theng Nga, Zhi Li Cheoka, Mohamad Aris Mohd Moklasb,
Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti
Putra Malaysia, Serdang, Selangor, Malaysia.
Department of Human Anatomy, Faculty of Medicine and Health Sciences, Universiti
AN US
b
ED
M
Putra Malaysia, Serdang, Selangor, Malaysia.
* Corresponding author.
PT
Zuraini Ahmad
CE
Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
AC
Phone number : +60389472313 E-mail address: [email protected]
1
ACCEPTED MANUSCRIPT
ABSTRACT Background: Endothelial cell activation is characterized by increased endothelial permeability and increased expression of cell adhesion molecules (CAMs). This allows monocyte adherence and migration across the endothelium to occur and thereby initiates atherogenesis process. Asiatic acid is a major triterpene isolated from Centella asiatica
CR IP T
(L.) Urban. and has been shown to possess anti-oxidant, anti-hyperlipidemia and antiinflammatory activities.
Purpose: We aimed to investigate protective effects of asiatic acid on tumor necrosis
factor-α (TNF-α)-induced endothelial cell activation using human aortic endothelial cells (HAECs).
AN US
Study design: For cell viability assays, HAECs were treated with asiatic acid for 24 h. For other assays, HAECs were pretreated with various doses of asiatic acid (10 – 40 μM) for 6 h followed by stimulation with TNF-α (10 ng/ml) for 6 h.
Methods: Fluorescein isothiocyanate (FITC)-dextran permeability assay was performed using commercial kits. Total protein expression of CAMs such as E-selectin, ICAM-1,
M
VCAM-1 and PECAM-1 as well as phosphorylation of IκB-α were determined using western blot. The levels of soluble form of CAMs were measured using flow cytometry.
ED
Besides, we also examined the effects of asiatic acid on U937 monocyte adhesion and monocyte migration in HAECs using fluorescent-based assays.
PT
Results: Asiatic acid significantly suppressed endothelial hyperpermeability, increased VCAM-1 expression and increased levels of soluble CAMs (sE-selectin, sICAM-1,
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sVCAM-1 and sPECAM-1) triggered by TNF-α. Neither TNF-α nor asiatic acid affects PECAM-1 expression. However, asiatic acid did not inhibit TNF-α-induced increased monocyte adhesion and migration. Interestingly, asiatic acid suppressed increased
AC
phosphorylation of IκB-α stimulated by TNF-α. Conclusion: These results suggest that asiatic acid protects against endothelial barrier disruption and this might be associated with the inhibition of NF-κB activation. We have demonstrated a novel protective role of asiatic acid on endothelial function. This reveals the possibility to further explore beneficial effects of asiatic acid on chronic inflammatory diseases that are initiated by endothelial cell activation.
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Keywords Asiatic acid; TNF-α; Human aortic endothelial cells; Cell adhesion molecules; NF-κB
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Abbreviations HAECs, human aortic endothelial cells; TNF-α, tumor necrosis factor-α; CAMs, cell adhesion molecules; sCAMs; soluble form of cell adhesion molecules; ICAM-1, intercellular adhesion molecule-1; VCAM-1, vascular cell adhesion molecule-1;
PECAM-1, platelet endothelial cell adhesion molecule-1; sE-selectin, soluble E-selectin;
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sICAM-1, soluble ICAM-1; sVCAM-1; soluble VCAM-1; sPECAM-1, soluble PECAM1; NF-κB, nuclear factor-κB; IκBα, inhibitor of NF-κB alpha; FITC-dextran, fluorescein isothiocyanate conjugated-dextran
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Introduction
Atherosclerosis is a chronic inflammatory disease initiated by endothelial dysfunction
ED
and ultimately develops into coronary heart disease. Hyperlipidemia, diabetes and hypertension are risk factors known to initiate atherogenesis. In early stage of
PT
atherosclerosis, endothelial dysfunction is accompanied by activation of endothelial cells that involves a complex interplay between leukocytes, endothelial cells and cytokines
CE
(Sitia et al., 2010). In response to pro-inflammatory stimuli, the surface expression of cell adhesion molecules (CAMs) such as E-selectin, intercellular adhesion molecule (ICAM)1 and vascular cell adhesion molecule (VCAM)-1 are up-regulated in endothelial cells.
AC
These favor the recruitment of circulating leukocytes and hence promote firm attachment between leukocytes and endothelial cells. In addition, ICAM-1 and VCAM-1 serve as signaling molecules that promote the production of reactive oxygen species (ROS), a key player of TNF-α-induced increased permeability (van Wetering et al., 2003; Wolf et al., 2013). Following the binding of monocytes to endothelium, platelet endothelial cell adhesion molecule (PECAM)-1 regulates the transmigration of monocytes across the blood vessel wall. Enzymatic cleavage of the surface CAMs results in the secretion of 3
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soluble form of cell adhesion molecules (sCAMs) such as soluble E-selectin (sE-selectin), soluble ICAM-1 (sICAM-1), soluble VCAM-1 (sVCAM-1) and soluble PECAM-1 (sPECAM-1) (Leeuwenberg et al., 1992). The level of sICAM-1 is elevated in hyperlipidemic individuals and associated with high cardiovascular risk (Karasek et al., 2005). Importantly, an increase in endothelial permeability to small molecules occurs in
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parallel with the diapedesis of monocytes, leading to disruption of endothelial barrier that subsequently ease the foam cell formation (Funk et al., 2012). Therefore, natural
compounds that protect against early atherogenic events that occur before the formation of atherosclerotic lesion might have beneficial effects in preventing atherosclerosis. Tumor necrosis factor-alpha (TNF-α), a pro-inflammatory cytokine, is found to be
AN US
expressed in atherosclerotic lesion (Barath et al., 1990). During the progression of
atherosclerosis, TNF-α sustains and propagates the inflammatory response by increasing the surface expression of CAMs, stimulating the production of inflammatory cytokines and chemokines as well as enhancing endothelial permeability. Although the molecular pathways that lead to TNF-α-induced increased permeability are not well understood,
M
accumulating evidence in recent years has suggested that the CAMs are central mechanisms in mediating endothelial hyperpermeability stimulated by TNF-α (Frank and
ED
Lisanti, 2008; Marcos-Ramiro et al., 2014). Nuclear factor-κB (NF-κB) family consists of a group of transcription factors that
PT
regulate various cellular processes such as inflammation, immune response and programmed cell death. In cytoplasm, the binding of these transcription factors with an
CE
inhibitory protein, inhibitor of NF-κB-alpha (IκBα), prevents them from entering the nucleus. Upon activation, IκBα phosphorylates and undergoes degradation while
AC
releasing the bound NF-κB dimer into the nucleus. The transcription cascades of many pro-inflammatory genes are then being initiated. Thus, inhibition of the NF-κB pathway might be a promising therapeutic strategy to prevent inflammatory diseases that are perpetuated by cytokines. Asiatic acid is one of the pentacyclic triterpenoids isolated from Centella asiatica (L.) Urban., a traditional medicinal plant that is commonly found in swampy areas of most tropical countries including India, China, Indonesia, Malaysia and other Asian countries. 4
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C. asiatica is well known for its wound healing and neuroprotective effects in Ayurvedic medicine. In Malaysia and Thailand, it is consumed as raw vegetables or blended into juice and served as tonic drinks (Hashim, 2011). The lipid lowering effects of C. asiatica extract were previously reported by other groups. C. asiatica extract was shown to reduce total cholesterol, triglyceride and plasma glucose in hyperlipidemic rats (Supkamonseni
CR IP T
et al., 2014). A fraction of ethanol extract of C. asiatica was reported to improve lipid profiles in chemical-induced hyperlipidemic mice and high fat diet induced-hamster models (Zhao et al., 2014). In several clinical studies, total triterpenic fraction of C.
asiatica (TTFCA) was demonstrated to prevent the progression of atherosclerotic plaques in asymptomatic patients when administered in combination with Pycnogenol, a pine
AN US
bark extract (Belcaro et al., 2015a; Belcaro et al., 2015b). In particular, TTFCA has been shown to improve capillary permeability in hypertensive patients, and this is associated with reduction of microcirculatory symptoms (Belcaro et al., 1990; De Sanctis et al., 2001). Anti-inflammatory, anti-angiogenesis and anti-oxidant effects of asiatic acid have also been reported previously (Huang et al., 2011; Kavitha et al., 2011; Pakdeechote et al.,
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2014). A study conducted using diabetic rats has reported that the ability of asiatic acid to reduce plasma glucose level might be associated with the hypolipidemic effect of asiatic
ED
acid (Ramachandran et al., 2014). Besides, asiatic acid also improves lipid profile of metabolic syndrome rats through maintaining the equilibrium between iNOS and eNOS
PT
expression (Pakdeechote et al., 2014). Besides, asiatic acid protects against high fat diet-induced liver injury in mice through
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inhibiting the NF-κB and mitogen-activated protein kinase (MAPK) pathways (Yan et al., 2014). These previous data suggest that asiatic acid possesses potential lipid lowering and anti-inflammatory effects. However, anti-atherosclerotic effects of asiatic acid and its
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underlying mechanisms are not well documented. Therefore, the aim of this study was to investigate the protective effects of asiatic acid on early atherogenic events, in the context of endothelial cell activation triggered by cytokines. We examined the in vitro effects of asiatic acid on TNF-α-induced increased endothelial permeability, expression of adhesion molecules, monocyte adhesion and monocyte migration. In addition, the effect of asiatic acid on NF-κB activation elicited by TNF-α was also explored in this study.
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Materials and methods Chemicals and reagents Asiatic acid was purchased from ChromaDex (CA, USA) with the purity of 93.7% (Supplementary material S1), 2’,7’-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein-
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acetoxymethyl ester (BCECF-AM) was purchased from Sigma (MO, USA). Rabbit polyclonal anti-ICAM-1 antibody, mouse monoclonal anti-E-selectin, anti-VCAM-1 and anti-PECAM-1 antibodies and anti-mouse IgG HRP-conjugated were purchased from Santa Cruz Biotechnology (Texas, USA). Rabbit monoclonal anti-phospho-IκBα
antibody and anti-rabbit HRP-conjugated secondary antibody were purchased from Cell
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Signaling Technology (MA, USA). TNF-α was purchased from Peprotech (NJ, USA).
Simvastatin and methyl thiazoyltetrazolium (MTT) were purchased from Calbiochem (NJ, USA). Cell culture
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Human aortic endothelial cells (HAECs) were purchased from American Type Cell Culture (ATCC) and maintained in endothelial cell medium (Sciencell, CA, USA)
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supplemented with 5% fetal bovine serum, endothelial cell growth supplement, penicillin (100 U/ml) and streptomycin (100 µg/ml). The medium was changed every two days until the cells were 80 - 90% confluent. All the experiments were performed using cells
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between passage 3 to 5. U937 cells, a suspension human leukemic monocyte lymphoma cell line, were purchased from ATCC and maintained in RPMI-1640 medium containing
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10% Hyclone fetal bovine serum (Thermo Fisher Scientific, IL, USA). The cell density was maintained at 1 x 105 to 2 x 106 cells per ml.
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For all the experiments except the cell viability assays, HAECs were pretreated with asiatic acid (10, 20, 30 and 40 μM) for 6 h before stimulated with TNF-α (10 ng/ml) for another 6 h. Cell viability assays Cell viability was assessed using MTT assay as described previously (Ng et al., 2015). HAECs were treated with various concentrations of asiatic acid (10 - 200 µM) for 24 h. 6
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After 24 h, we added 10 µl of MTT solution (5 mg/ml in PBS) into each well and incubated for 4 h. Then, all the solution was removed and 100 µl of DMSO was added to dissolve the purple formazan salt formed. The absorbance was read at 570 nm with a reference wavelength of 650 nm. In addition, cell viability was also assessed by a fluorometric-based assay. ATP fluorometric assay kit (Biovision, CA, USA) was used to
excitation/emission wavelengths of 535 nm/587 nm. Permeability assay in vitro
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quantify the ATP level in viable cells. Fluorescence intensity was read at
In vitro Vascular Permeability Assay kit (Milipore, MA, USA) was used to measure the
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passage of fluorescein isothiocyanate (FITC) conjugated-dextran across HAECs
monolayer according to manufacturer’s protocol. HAECs were grown in collagen-coated cell culture inserts for 3 days to allow the formation of cell monolayers. The inserts were placed in a 24-well plate where the bottom wells were filled with 500 µl of endothelial cell medium. After treatment, 150 µl of FITC- dextran was added to the inserts and
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incubated for 20 min. At the end of experiment, 100 µl of media was collected from the bottom wells and transferred to a black 96-well plate. Fluorescence intensity was
ED
measured at wavelengths of 485 nm excitation and 535 nm emission using a fluorescence microplate reader (Infinite M200, TECAN, Männedorf, Switzerland). Results are expressed as a percentage compared to control (Relative fluorescence unit fluorescence unitcontrol x 100%).
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treatment/Relative
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Measurement of soluble CAMs in cell culture supernatant The concentrations of sE-selectin, sP-selectin, sICAM-1, sVCAM-1 and s-PECAM-1 in
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cell culture supernatant were measured by using human adhesion 6plex kit (eBioscience, Vienna, Austria) according to the manufacturer’s protocol. The fluorescent intensity of samples was acquired using BD FACS Calibur flow cytometer (BD Biosciences, NJ, USA). Standards were run in parallel with samples for each independent experiment and the concentrations of samples were obtained from the standard curve.
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Fluorescence labeling of U937 monocytes U937 monocytes at a concentration of 3 x 106 cells per ml were fluorescently labeled with 2 µM of BCECF-AM in RPMI-1640 medium for 45 min at 37 oC and 5% CO2. The
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monocytes were washed twice with PBS containing 0.5% BSA to remove unbound dye before resuspended in endothelial cell medium. BCECF-AM-labeled U937 monocytes were then used for monocyte adhesion and migration assays. U937 monocyte adhesion assay
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U937 monocyte adhesion assay was carried out according to the procedures previously described with some modifications (Ang et al., 2011). BCECF-AM-labeled monocytes with a density of 1 x 105 cells per well were added onto the monolayers of HAECs and incubated for 30 min at 37 oC and 5% CO2. The monolayers were washed three times
with PBS to remove non-adhering U937 monocytes. The attached monocytes were then
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lysed with 0.1% Triton X-100 in 0.1M NaOH. The fluorescence intensities were measured at excitation and emission wavelengths of 485 nm and 535 nm, respectively.
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The fluorescence intensities of known numbers of labeled monocytes were also measured and used to construct a standard curve for each set of experiment. The number of attached monocytes for each group was calculated from the standard curve. Results are expressed
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as a percentage compared to normal control. For qualitative analysis, HAECs were grown onto 22 mm collagen-coated coverslips (BD Bioscience, NJ, USA). At the end of
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treatment, BCECF-AM-labeled monocytes were added to the HAECs and non-binding monocytes were removed by gently washing the cell monolayer with PBS. Then, the cells
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were fixed in 3.7% paraformaldehyde and mounted with ProLong Gold antifade agent (Molecular Probes, OR, USA). 5 random fields were captured for each independent experiment using a Leica DM2500 fluorescence microscope (Leica Microsystems Vertrieb GmbH, Wetzlar, Germany). Migration assay in vitro
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A fluorescent-based transendothelial migration assay was performed as described (Ramirez et al., 2008) with some modifications. Cell culture inserts with the pore size of 3 µm (BD Biosciences, NJ, USA) were coated with rat tail collagen type I (BD Biosciences, NJ, USA). HAECs were grown onto the collagen-coated inserts at a density of 2 x 105 cells per insert for three days to allow confluent monolayers to be formed.
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After treatment, the BCECF-AM-labeled U937 monocytes were added to the upper chamber and incubated at 37 oC for 2 h to allow transmigration of monocytes to the lower chamber. Media from the lower chamber was collected and transferred to a black opaque 96-well plate for the measurement of fluorescence intensity using a fluorescence
microplate reader (Infinite M200, TECAN, Männedorf, Switzerland) at excitation and
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emission wavelengths of 485 nm and 535 nm, respectively. The fluorescence intensities for known numbers of labeled monocytes were used to plot standard curves and the number of migrated monocytes was calculated from the curve. Western blot analysis
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The cells were lysed in RIPA buffer. The supernatant was collected and the protein concentrations were quantified using bicinchoninic acid (BCA) protein assay reagent kit
ED
(Pierce, Rockford, USA). Equal amount of proteins were loaded onto 10-15% polyacrylamide gel and resolved by SDS-PAGE. The proteins were transferred onto a PVDF membrane, blocked in 5% BSA for 1 h and incubated with the following primary
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antibodies; ICAM-1 (1:3000 dilution), VCAM-1 (1:1000 dilution), E-selectin (1:2000 dilution), PECAM-1 (1 : 3000 dilution), phospho-IκBα (1:1000 dilution). The membranes
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were washed three times, 5 min each, and incubated with HRP-linked secondary antibody (1:5000 dilution). Chemiluminescent signal was developed using LuminataTM Forte
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Western HRP substrate (Milipore, MA, USA). Densitometry analysis was performed using Image J software. Statistical analysis The results are expressed as the mean ± standard mean of error (SEM). All the data were analyzed with one way analysis of variance (ANOVA) followed by Dunnett’s test. P < 0.05 was considered as statistically significant. 9
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Results
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Asiatic acid does not decrease viability of HAECs In order to ensure all the concentrations of asiatic acid used in this study does not affect the viability of HAECs, MTT assay was performed by incubating the cells with various concentrations of asiatic acid for 24 h. Asiatic acid, when applied up to 40 µM, did not cause significant cell death (Fig. 1A). These results are further confirmed with a more
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sensitive fluorometric assay that measures the ATP level in viable cells. We obtained
similar results as MTT assay where 40 µM of asiatic acid did not significantly reduce the amount of ATP compared to control cells (Fig. 1B). Therefore, 10 - 40 µM were chosen as the treatment dosages in this study. Taken together, the inhibitory effects of asiatic acid in all the subsequent experiments were not due to cytotoxic effect in HAECs.
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Asiatic acid suppresses TNF-α-induced endothelial barrier disruption in HAECs
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The amount of FITC-labeled dextran that passed through the HAECs monolayer was used to indicate the integrity of endothelial barrier. As shown in Fig. 2, TNF-α significantly impaired the barrier integrity and caused an increase in permeability to
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156.8 ± 4.8 % of control. Asiatic acid significantly abolished the TNF-α-induced increased permeability in a dose-dependent manner (P < 0.05). However, asiatic acid
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alone did not alter the baseline permeability. We used simvastatin, a drug that is commonly used in clinical practice to prevent atherosclerosis, as a positive control for our
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assays. Simvastatin significantly decreased the hyperpermeability stimulated by TNF-α to 104.9 ± 3.45 % of control (Fig. 2). These results indicate that asiatic acid maintains the barrier integrity and prevents the barrier disruption induced by TNF-α. Asiatic acid decreases TNF-α-induced increased release of soluble CAMs Non-activated HAECs released sVCAM-1 and sICAM-1 at 2.67 ± 0.01 ng/ml and 9.30 ± 1.78 ng/ml, respectively; while sPECAM-1 was secreted at a relatively higher level, 21.9 10
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± 0.96 ng/ml (Fig. 3). Non-activated HAECs did not secrete sE-selectin. Upon stimulation with 10 ng/ml of TNF-α, the concentrations of sE-selectin, sICAM-1, sVCAM-1 and sPECAM-1 in the cell culture supernatant were significantly increased to 176.6 ± 31.94, 191.5 ± 7.16, 39.78 ± 0.34 and 272 ± 16.62 ng/ml, respectively (P < 0.05). Asiatic acid significantly reduced the sE-selectin, sICAM-1 and sPECAM-1 levels at all
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doses tested (P < 0.05) (Fig. 3). The suppressive effects were not dose dependent. Asiatic acid also significantly suppressed increased sVCAM-1 level at 20 - 40 µM in a dose
dependent manner (P < 0.05). Furthermore, the concentrations of all sCAMs were not
significantly different from control group when cells were treated with asiatic acid alone (40 µM). As shown in Fig. 3, simvastatin reduced TNF-α-induced protein secretion of
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sE-selectin, sICAM-1, sVCAM-1 and sPECAM-1 (P < 0.05). In summary, asiatic acid
inhibits the secretion of sCAMs, which are biological markers used in the risk prediction of cardiovascular diseases.
Asiatic acid inhibits increased total protein expression of VCAM-1 triggered by TNF-α
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In order to investigate whether asiatic acid could also suppress total protein expression of CAMs, total expression of E-selectin, ICAM-1, VCAM-1 and PECAM-1 were detected
ED
using western blot. Non-stimulated HAECs did not express E-selectin and VCAM-1; while ICAM-1 and PECAM-1 were expressed (Fig. 4A). When cells were induced with TNF-α, E-selectin, ICAM-1 and VCAM-1 expression were significantly increased (P
Barrier protective effect of asiatic acid in TNF-α-induced activation of human aortic endothelial cells Lai Yen Fong , Chin Theng Ng , Zhi Li Cheok , Mohamad Aris Mohd Moklas , Muhammad Nazrul Hakim , Zuraini Ahmad PII: DOI: Reference:
S0944-7113(15)00384-0 10.1016/j.phymed.2015.11.019 PHYMED 51943
To appear in:
Phytomedicine
Received date: Revised date: Accepted date:
14 October 2015 17 November 2015 26 November 2015
Please cite this article as: Lai Yen Fong , Chin Theng Ng , Zhi Li Cheok , Mohamad Aris Mohd Moklas , Muhammad Nazrul Hakim , Zuraini Ahmad , Barrier protective effect of asiatic acid in TNF-α-induced activation of human aortic endothelial cells, Phytomedicine (2016), doi: 10.1016/j.phymed.2015.11.019
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Barrier protective effect of asiatic acid in TNF-α-induced activation of human aortic endothelial cells
Muhammad Nazrul Hakima and Zuraini Ahmada,*
a
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Lai Yen Fonga, Chin Theng Nga, Zhi Li Cheoka, Mohamad Aris Mohd Moklasb,
Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti
Putra Malaysia, Serdang, Selangor, Malaysia.
Department of Human Anatomy, Faculty of Medicine and Health Sciences, Universiti
AN US
b
ED
M
Putra Malaysia, Serdang, Selangor, Malaysia.
* Corresponding author.
PT
Zuraini Ahmad
CE
Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
AC
Phone number : +60389472313 E-mail address: [email protected]
1
ACCEPTED MANUSCRIPT
ABSTRACT Background: Endothelial cell activation is characterized by increased endothelial permeability and increased expression of cell adhesion molecules (CAMs). This allows monocyte adherence and migration across the endothelium to occur and thereby initiates atherogenesis process. Asiatic acid is a major triterpene isolated from Centella asiatica
CR IP T
(L.) Urban. and has been shown to possess anti-oxidant, anti-hyperlipidemia and antiinflammatory activities.
Purpose: We aimed to investigate protective effects of asiatic acid on tumor necrosis
factor-α (TNF-α)-induced endothelial cell activation using human aortic endothelial cells (HAECs).
AN US
Study design: For cell viability assays, HAECs were treated with asiatic acid for 24 h. For other assays, HAECs were pretreated with various doses of asiatic acid (10 – 40 μM) for 6 h followed by stimulation with TNF-α (10 ng/ml) for 6 h.
Methods: Fluorescein isothiocyanate (FITC)-dextran permeability assay was performed using commercial kits. Total protein expression of CAMs such as E-selectin, ICAM-1,
M
VCAM-1 and PECAM-1 as well as phosphorylation of IκB-α were determined using western blot. The levels of soluble form of CAMs were measured using flow cytometry.
ED
Besides, we also examined the effects of asiatic acid on U937 monocyte adhesion and monocyte migration in HAECs using fluorescent-based assays.
PT
Results: Asiatic acid significantly suppressed endothelial hyperpermeability, increased VCAM-1 expression and increased levels of soluble CAMs (sE-selectin, sICAM-1,
CE
sVCAM-1 and sPECAM-1) triggered by TNF-α. Neither TNF-α nor asiatic acid affects PECAM-1 expression. However, asiatic acid did not inhibit TNF-α-induced increased monocyte adhesion and migration. Interestingly, asiatic acid suppressed increased
AC
phosphorylation of IκB-α stimulated by TNF-α. Conclusion: These results suggest that asiatic acid protects against endothelial barrier disruption and this might be associated with the inhibition of NF-κB activation. We have demonstrated a novel protective role of asiatic acid on endothelial function. This reveals the possibility to further explore beneficial effects of asiatic acid on chronic inflammatory diseases that are initiated by endothelial cell activation.
2
ACCEPTED MANUSCRIPT
Keywords Asiatic acid; TNF-α; Human aortic endothelial cells; Cell adhesion molecules; NF-κB
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Abbreviations HAECs, human aortic endothelial cells; TNF-α, tumor necrosis factor-α; CAMs, cell adhesion molecules; sCAMs; soluble form of cell adhesion molecules; ICAM-1, intercellular adhesion molecule-1; VCAM-1, vascular cell adhesion molecule-1;
PECAM-1, platelet endothelial cell adhesion molecule-1; sE-selectin, soluble E-selectin;
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sICAM-1, soluble ICAM-1; sVCAM-1; soluble VCAM-1; sPECAM-1, soluble PECAM1; NF-κB, nuclear factor-κB; IκBα, inhibitor of NF-κB alpha; FITC-dextran, fluorescein isothiocyanate conjugated-dextran
M
Introduction
Atherosclerosis is a chronic inflammatory disease initiated by endothelial dysfunction
ED
and ultimately develops into coronary heart disease. Hyperlipidemia, diabetes and hypertension are risk factors known to initiate atherogenesis. In early stage of
PT
atherosclerosis, endothelial dysfunction is accompanied by activation of endothelial cells that involves a complex interplay between leukocytes, endothelial cells and cytokines
CE
(Sitia et al., 2010). In response to pro-inflammatory stimuli, the surface expression of cell adhesion molecules (CAMs) such as E-selectin, intercellular adhesion molecule (ICAM)1 and vascular cell adhesion molecule (VCAM)-1 are up-regulated in endothelial cells.
AC
These favor the recruitment of circulating leukocytes and hence promote firm attachment between leukocytes and endothelial cells. In addition, ICAM-1 and VCAM-1 serve as signaling molecules that promote the production of reactive oxygen species (ROS), a key player of TNF-α-induced increased permeability (van Wetering et al., 2003; Wolf et al., 2013). Following the binding of monocytes to endothelium, platelet endothelial cell adhesion molecule (PECAM)-1 regulates the transmigration of monocytes across the blood vessel wall. Enzymatic cleavage of the surface CAMs results in the secretion of 3
ACCEPTED MANUSCRIPT
soluble form of cell adhesion molecules (sCAMs) such as soluble E-selectin (sE-selectin), soluble ICAM-1 (sICAM-1), soluble VCAM-1 (sVCAM-1) and soluble PECAM-1 (sPECAM-1) (Leeuwenberg et al., 1992). The level of sICAM-1 is elevated in hyperlipidemic individuals and associated with high cardiovascular risk (Karasek et al., 2005). Importantly, an increase in endothelial permeability to small molecules occurs in
CR IP T
parallel with the diapedesis of monocytes, leading to disruption of endothelial barrier that subsequently ease the foam cell formation (Funk et al., 2012). Therefore, natural
compounds that protect against early atherogenic events that occur before the formation of atherosclerotic lesion might have beneficial effects in preventing atherosclerosis. Tumor necrosis factor-alpha (TNF-α), a pro-inflammatory cytokine, is found to be
AN US
expressed in atherosclerotic lesion (Barath et al., 1990). During the progression of
atherosclerosis, TNF-α sustains and propagates the inflammatory response by increasing the surface expression of CAMs, stimulating the production of inflammatory cytokines and chemokines as well as enhancing endothelial permeability. Although the molecular pathways that lead to TNF-α-induced increased permeability are not well understood,
M
accumulating evidence in recent years has suggested that the CAMs are central mechanisms in mediating endothelial hyperpermeability stimulated by TNF-α (Frank and
ED
Lisanti, 2008; Marcos-Ramiro et al., 2014). Nuclear factor-κB (NF-κB) family consists of a group of transcription factors that
PT
regulate various cellular processes such as inflammation, immune response and programmed cell death. In cytoplasm, the binding of these transcription factors with an
CE
inhibitory protein, inhibitor of NF-κB-alpha (IκBα), prevents them from entering the nucleus. Upon activation, IκBα phosphorylates and undergoes degradation while
AC
releasing the bound NF-κB dimer into the nucleus. The transcription cascades of many pro-inflammatory genes are then being initiated. Thus, inhibition of the NF-κB pathway might be a promising therapeutic strategy to prevent inflammatory diseases that are perpetuated by cytokines. Asiatic acid is one of the pentacyclic triterpenoids isolated from Centella asiatica (L.) Urban., a traditional medicinal plant that is commonly found in swampy areas of most tropical countries including India, China, Indonesia, Malaysia and other Asian countries. 4
ACCEPTED MANUSCRIPT
C. asiatica is well known for its wound healing and neuroprotective effects in Ayurvedic medicine. In Malaysia and Thailand, it is consumed as raw vegetables or blended into juice and served as tonic drinks (Hashim, 2011). The lipid lowering effects of C. asiatica extract were previously reported by other groups. C. asiatica extract was shown to reduce total cholesterol, triglyceride and plasma glucose in hyperlipidemic rats (Supkamonseni
CR IP T
et al., 2014). A fraction of ethanol extract of C. asiatica was reported to improve lipid profiles in chemical-induced hyperlipidemic mice and high fat diet induced-hamster models (Zhao et al., 2014). In several clinical studies, total triterpenic fraction of C.
asiatica (TTFCA) was demonstrated to prevent the progression of atherosclerotic plaques in asymptomatic patients when administered in combination with Pycnogenol, a pine
AN US
bark extract (Belcaro et al., 2015a; Belcaro et al., 2015b). In particular, TTFCA has been shown to improve capillary permeability in hypertensive patients, and this is associated with reduction of microcirculatory symptoms (Belcaro et al., 1990; De Sanctis et al., 2001). Anti-inflammatory, anti-angiogenesis and anti-oxidant effects of asiatic acid have also been reported previously (Huang et al., 2011; Kavitha et al., 2011; Pakdeechote et al.,
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2014). A study conducted using diabetic rats has reported that the ability of asiatic acid to reduce plasma glucose level might be associated with the hypolipidemic effect of asiatic
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acid (Ramachandran et al., 2014). Besides, asiatic acid also improves lipid profile of metabolic syndrome rats through maintaining the equilibrium between iNOS and eNOS
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expression (Pakdeechote et al., 2014). Besides, asiatic acid protects against high fat diet-induced liver injury in mice through
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inhibiting the NF-κB and mitogen-activated protein kinase (MAPK) pathways (Yan et al., 2014). These previous data suggest that asiatic acid possesses potential lipid lowering and anti-inflammatory effects. However, anti-atherosclerotic effects of asiatic acid and its
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underlying mechanisms are not well documented. Therefore, the aim of this study was to investigate the protective effects of asiatic acid on early atherogenic events, in the context of endothelial cell activation triggered by cytokines. We examined the in vitro effects of asiatic acid on TNF-α-induced increased endothelial permeability, expression of adhesion molecules, monocyte adhesion and monocyte migration. In addition, the effect of asiatic acid on NF-κB activation elicited by TNF-α was also explored in this study.
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Materials and methods Chemicals and reagents Asiatic acid was purchased from ChromaDex (CA, USA) with the purity of 93.7% (Supplementary material S1), 2’,7’-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein-
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acetoxymethyl ester (BCECF-AM) was purchased from Sigma (MO, USA). Rabbit polyclonal anti-ICAM-1 antibody, mouse monoclonal anti-E-selectin, anti-VCAM-1 and anti-PECAM-1 antibodies and anti-mouse IgG HRP-conjugated were purchased from Santa Cruz Biotechnology (Texas, USA). Rabbit monoclonal anti-phospho-IκBα
antibody and anti-rabbit HRP-conjugated secondary antibody were purchased from Cell
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Signaling Technology (MA, USA). TNF-α was purchased from Peprotech (NJ, USA).
Simvastatin and methyl thiazoyltetrazolium (MTT) were purchased from Calbiochem (NJ, USA). Cell culture
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Human aortic endothelial cells (HAECs) were purchased from American Type Cell Culture (ATCC) and maintained in endothelial cell medium (Sciencell, CA, USA)
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supplemented with 5% fetal bovine serum, endothelial cell growth supplement, penicillin (100 U/ml) and streptomycin (100 µg/ml). The medium was changed every two days until the cells were 80 - 90% confluent. All the experiments were performed using cells
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between passage 3 to 5. U937 cells, a suspension human leukemic monocyte lymphoma cell line, were purchased from ATCC and maintained in RPMI-1640 medium containing
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10% Hyclone fetal bovine serum (Thermo Fisher Scientific, IL, USA). The cell density was maintained at 1 x 105 to 2 x 106 cells per ml.
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For all the experiments except the cell viability assays, HAECs were pretreated with asiatic acid (10, 20, 30 and 40 μM) for 6 h before stimulated with TNF-α (10 ng/ml) for another 6 h. Cell viability assays Cell viability was assessed using MTT assay as described previously (Ng et al., 2015). HAECs were treated with various concentrations of asiatic acid (10 - 200 µM) for 24 h. 6
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After 24 h, we added 10 µl of MTT solution (5 mg/ml in PBS) into each well and incubated for 4 h. Then, all the solution was removed and 100 µl of DMSO was added to dissolve the purple formazan salt formed. The absorbance was read at 570 nm with a reference wavelength of 650 nm. In addition, cell viability was also assessed by a fluorometric-based assay. ATP fluorometric assay kit (Biovision, CA, USA) was used to
excitation/emission wavelengths of 535 nm/587 nm. Permeability assay in vitro
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quantify the ATP level in viable cells. Fluorescence intensity was read at
In vitro Vascular Permeability Assay kit (Milipore, MA, USA) was used to measure the
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passage of fluorescein isothiocyanate (FITC) conjugated-dextran across HAECs
monolayer according to manufacturer’s protocol. HAECs were grown in collagen-coated cell culture inserts for 3 days to allow the formation of cell monolayers. The inserts were placed in a 24-well plate where the bottom wells were filled with 500 µl of endothelial cell medium. After treatment, 150 µl of FITC- dextran was added to the inserts and
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incubated for 20 min. At the end of experiment, 100 µl of media was collected from the bottom wells and transferred to a black 96-well plate. Fluorescence intensity was
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measured at wavelengths of 485 nm excitation and 535 nm emission using a fluorescence microplate reader (Infinite M200, TECAN, Männedorf, Switzerland). Results are expressed as a percentage compared to control (Relative fluorescence unit fluorescence unitcontrol x 100%).
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treatment/Relative
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Measurement of soluble CAMs in cell culture supernatant The concentrations of sE-selectin, sP-selectin, sICAM-1, sVCAM-1 and s-PECAM-1 in
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cell culture supernatant were measured by using human adhesion 6plex kit (eBioscience, Vienna, Austria) according to the manufacturer’s protocol. The fluorescent intensity of samples was acquired using BD FACS Calibur flow cytometer (BD Biosciences, NJ, USA). Standards were run in parallel with samples for each independent experiment and the concentrations of samples were obtained from the standard curve.
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Fluorescence labeling of U937 monocytes U937 monocytes at a concentration of 3 x 106 cells per ml were fluorescently labeled with 2 µM of BCECF-AM in RPMI-1640 medium for 45 min at 37 oC and 5% CO2. The
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monocytes were washed twice with PBS containing 0.5% BSA to remove unbound dye before resuspended in endothelial cell medium. BCECF-AM-labeled U937 monocytes were then used for monocyte adhesion and migration assays. U937 monocyte adhesion assay
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U937 monocyte adhesion assay was carried out according to the procedures previously described with some modifications (Ang et al., 2011). BCECF-AM-labeled monocytes with a density of 1 x 105 cells per well were added onto the monolayers of HAECs and incubated for 30 min at 37 oC and 5% CO2. The monolayers were washed three times
with PBS to remove non-adhering U937 monocytes. The attached monocytes were then
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lysed with 0.1% Triton X-100 in 0.1M NaOH. The fluorescence intensities were measured at excitation and emission wavelengths of 485 nm and 535 nm, respectively.
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The fluorescence intensities of known numbers of labeled monocytes were also measured and used to construct a standard curve for each set of experiment. The number of attached monocytes for each group was calculated from the standard curve. Results are expressed
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as a percentage compared to normal control. For qualitative analysis, HAECs were grown onto 22 mm collagen-coated coverslips (BD Bioscience, NJ, USA). At the end of
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treatment, BCECF-AM-labeled monocytes were added to the HAECs and non-binding monocytes were removed by gently washing the cell monolayer with PBS. Then, the cells
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were fixed in 3.7% paraformaldehyde and mounted with ProLong Gold antifade agent (Molecular Probes, OR, USA). 5 random fields were captured for each independent experiment using a Leica DM2500 fluorescence microscope (Leica Microsystems Vertrieb GmbH, Wetzlar, Germany). Migration assay in vitro
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A fluorescent-based transendothelial migration assay was performed as described (Ramirez et al., 2008) with some modifications. Cell culture inserts with the pore size of 3 µm (BD Biosciences, NJ, USA) were coated with rat tail collagen type I (BD Biosciences, NJ, USA). HAECs were grown onto the collagen-coated inserts at a density of 2 x 105 cells per insert for three days to allow confluent monolayers to be formed.
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After treatment, the BCECF-AM-labeled U937 monocytes were added to the upper chamber and incubated at 37 oC for 2 h to allow transmigration of monocytes to the lower chamber. Media from the lower chamber was collected and transferred to a black opaque 96-well plate for the measurement of fluorescence intensity using a fluorescence
microplate reader (Infinite M200, TECAN, Männedorf, Switzerland) at excitation and
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emission wavelengths of 485 nm and 535 nm, respectively. The fluorescence intensities for known numbers of labeled monocytes were used to plot standard curves and the number of migrated monocytes was calculated from the curve. Western blot analysis
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The cells were lysed in RIPA buffer. The supernatant was collected and the protein concentrations were quantified using bicinchoninic acid (BCA) protein assay reagent kit
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(Pierce, Rockford, USA). Equal amount of proteins were loaded onto 10-15% polyacrylamide gel and resolved by SDS-PAGE. The proteins were transferred onto a PVDF membrane, blocked in 5% BSA for 1 h and incubated with the following primary
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antibodies; ICAM-1 (1:3000 dilution), VCAM-1 (1:1000 dilution), E-selectin (1:2000 dilution), PECAM-1 (1 : 3000 dilution), phospho-IκBα (1:1000 dilution). The membranes
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were washed three times, 5 min each, and incubated with HRP-linked secondary antibody (1:5000 dilution). Chemiluminescent signal was developed using LuminataTM Forte
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Western HRP substrate (Milipore, MA, USA). Densitometry analysis was performed using Image J software. Statistical analysis The results are expressed as the mean ± standard mean of error (SEM). All the data were analyzed with one way analysis of variance (ANOVA) followed by Dunnett’s test. P < 0.05 was considered as statistically significant. 9
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Results
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Asiatic acid does not decrease viability of HAECs In order to ensure all the concentrations of asiatic acid used in this study does not affect the viability of HAECs, MTT assay was performed by incubating the cells with various concentrations of asiatic acid for 24 h. Asiatic acid, when applied up to 40 µM, did not cause significant cell death (Fig. 1A). These results are further confirmed with a more
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sensitive fluorometric assay that measures the ATP level in viable cells. We obtained
similar results as MTT assay where 40 µM of asiatic acid did not significantly reduce the amount of ATP compared to control cells (Fig. 1B). Therefore, 10 - 40 µM were chosen as the treatment dosages in this study. Taken together, the inhibitory effects of asiatic acid in all the subsequent experiments were not due to cytotoxic effect in HAECs.
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Asiatic acid suppresses TNF-α-induced endothelial barrier disruption in HAECs
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The amount of FITC-labeled dextran that passed through the HAECs monolayer was used to indicate the integrity of endothelial barrier. As shown in Fig. 2, TNF-α significantly impaired the barrier integrity and caused an increase in permeability to
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156.8 ± 4.8 % of control. Asiatic acid significantly abolished the TNF-α-induced increased permeability in a dose-dependent manner (P < 0.05). However, asiatic acid
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alone did not alter the baseline permeability. We used simvastatin, a drug that is commonly used in clinical practice to prevent atherosclerosis, as a positive control for our
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assays. Simvastatin significantly decreased the hyperpermeability stimulated by TNF-α to 104.9 ± 3.45 % of control (Fig. 2). These results indicate that asiatic acid maintains the barrier integrity and prevents the barrier disruption induced by TNF-α. Asiatic acid decreases TNF-α-induced increased release of soluble CAMs Non-activated HAECs released sVCAM-1 and sICAM-1 at 2.67 ± 0.01 ng/ml and 9.30 ± 1.78 ng/ml, respectively; while sPECAM-1 was secreted at a relatively higher level, 21.9 10
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± 0.96 ng/ml (Fig. 3). Non-activated HAECs did not secrete sE-selectin. Upon stimulation with 10 ng/ml of TNF-α, the concentrations of sE-selectin, sICAM-1, sVCAM-1 and sPECAM-1 in the cell culture supernatant were significantly increased to 176.6 ± 31.94, 191.5 ± 7.16, 39.78 ± 0.34 and 272 ± 16.62 ng/ml, respectively (P < 0.05). Asiatic acid significantly reduced the sE-selectin, sICAM-1 and sPECAM-1 levels at all
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doses tested (P < 0.05) (Fig. 3). The suppressive effects were not dose dependent. Asiatic acid also significantly suppressed increased sVCAM-1 level at 20 - 40 µM in a dose
dependent manner (P < 0.05). Furthermore, the concentrations of all sCAMs were not
significantly different from control group when cells were treated with asiatic acid alone (40 µM). As shown in Fig. 3, simvastatin reduced TNF-α-induced protein secretion of
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sE-selectin, sICAM-1, sVCAM-1 and sPECAM-1 (P < 0.05). In summary, asiatic acid
inhibits the secretion of sCAMs, which are biological markers used in the risk prediction of cardiovascular diseases.
Asiatic acid inhibits increased total protein expression of VCAM-1 triggered by TNF-α
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In order to investigate whether asiatic acid could also suppress total protein expression of CAMs, total expression of E-selectin, ICAM-1, VCAM-1 and PECAM-1 were detected
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using western blot. Non-stimulated HAECs did not express E-selectin and VCAM-1; while ICAM-1 and PECAM-1 were expressed (Fig. 4A). When cells were induced with TNF-α, E-selectin, ICAM-1 and VCAM-1 expression were significantly increased (P
