Chlorella 11-Peptide Inhibits the Production of Macrophage-Induced Adhesion Molecules and Reduces Endothelin-1 Expression and Endothelial Permeability

The inflammation process in large vessels involves the up-regulation of vascular adhesion molecules such as endothelial cell selectin (E-selectin), intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) which are also known as the markers of atherosclerosis. We have reported that Chlorella 11-peptide exhibited effective anti-inflammatory effects. This peptide with an amino sequence Val-Glu-Cys-Tyr-Gly-Pro-Asn-Arg-Pro-Gln-Phe was further examined for its potential in preventing atherosclerosis in this study. In particular, the roles of Chlorella 11-peptide in lowering the production of vascular adhesion molecules, monocyte chemoattractant protein (MCP-1) and expression of endothelin-1 (ET-1) from endothelia (SVEC4-10 cells) were studied. The production of E-selectin, ICAM-1, VCAM-1 and MCP-1 in SVEC4-10 cells was measured with ELISA. The mRNA expression of ET-1 was analyzed by RT-PCR and agarose gel. Results showed that Chlorella 11-peptide significantly suppressed the levels of E-selectin, ICAM, VCAM, MCP-1 as well as ET-1 gene expression. The inhibition of ICAM-1 and VCAM-1 production by Chlorella 11-peptide was reversed in the presence of protein kinase A inhibitor (H89) which suggests that the cAMP pathway was involved in the inhibitory cause of the peptide. In addition, this peptide was shown to reduce the extent of increased intercellular permeability induced by combination of 50% of lipopolysaccharide (LPS)-activated RAW 264.7 cells medium and 50% normal SEVC cell culture medium (referred to as 50% RAW-conditioned medium). These data demonstrate that Chlorella 11-peptide is a promising biomolecule in preventing chronic inflammatory-related vascular diseases.


Introduction
Circulating adhesion molecules (CAMs) are proteins expressed by vascular endothelium that are believed to play a role in the initiation of the atherosclerotic process.There are several families of CAMs, including integrins, cadherins, selectins and immunoglobulin superfamily members [1].However, the most important adhesion molecules involved in atherosclerosis appear to be intercellular cell adhesion molecule-1 (ICAM-1), endothelial cell selectin (E-selectin), and vascular cell adhesion molecule-1 (VCAM-1).An elevated level of ICAM-1 has shown to correlate to an increased risk of cardiovascular events [2].E-selectin levels correlate with cardiovascular disease (CVD) risk [3] and have been used successfully to predict the severity of atherosclerosis in patients [4].VCAM-1 is a specific marker for advanced atherosclerosis, since it is often expressed in atherosclerotic plaques [5].A high level of VCAM-1 is usually associated with an increased risk of coronary events in persons with existing CVD [2].Studies also indicate that reducing the circulating level of these adhesion molecules could lower the CVD risk [6].Up-regulation of the adhesion molecules on endothelial cells is prominent after these cells are exposed to pro-inflammatory molecules such as tumor necrosis factor (TNF)-α, interleukin (IL)-8, and monocyte chemoattractant protein-1 (MCP-1) [7].Initiated by MCP-1, the recruitment and activation of monocytes/macrophages are followed and contribute to the initiation and pathophysiology of ischemic heart disease [8,9].In addition, MCP-1 is believed to be involved in the development of atherosclerosis [10], coronary artery disease [11], postischemic myocardial remodeling [12], and heart failure [13].Research on anti-MCP-1 gene therapy has successfully gained the attenuation of atherosclerosis in ApoE-deficient mice [14], suggesting MCP-1 as a potential therapeutic target in atherosclerosis.
Furthermore, endothelin-1 (ET-1) secreted by vascular endothelial cells acts as a potent endogenous vasoconstrictor and has an important role in the etiology of atherosclerotic vascular disease [15].When endothelia are under inflammation, their increased intercellular permeability would also permit cholesterol uptake within the vessel wall [15].Therefore, reducing the elevated ET-1 expression and remaining endothelial intercellular permeability to normal has been demonstrated to be an effective approach to block the development of circulatory disorders, including hypertension and atherosclerosis [16].
Green algae are able to prevent hyperlipidemia induced by a high fat diet [17] and atherosclerosis in laboratory animals [18].Additionally, Chlorella 11-peptide derived from the green algae demonstrates various biological effects.The components of the Chlorella 11-peptide are (Val-Glu-Cys-Tyr-Gly-Pro-Asn-Arg-Pro-Gln-Phe).It has recently been shown to decrease inducible nitric oxide synthase (iNOS) expression and Nuclear factor-kappa-B (NF-κB) activity and to possess antioxidant properties [19,20].Since atherosclerosis is known to be associated with an elevated cholesterol and chronic inflammation [21], examination of the relations between adhesion molecules, MCP-1, ET-1, endothelium membrane integrity with Chlorella 11-peptide would help to explore their therapeutic potential on atherosclerosis.In this study, the supernatant of LPS-stimulated RAW264.7 culture was added to endothelial cells (SVEC4-10) for stimulation of these cells.This is because activation of macrophage produces pro-inflammatory cytokines required for the development of atherosclerosis [22].After the stimuli to SVEC4-10, responses of endothelia with and without Chlorella 11-peptide were then evaluated.

Inhibitory Effects of Chlorella 11-Peptide on LPS-Induced MCP-1 Production in RAW264.7 Macrophages
The difference of MCP-1 production between LPS-treated and basal groups reached a maximum at 12 h after LPS stimulation [23].Therefore, this time point was chosen for further study (p < 0.005, Figure 1).The LPS-induced MCP-1 production was significantly inhibited (p < 0.005 and p < 0.05) by Chlorella 11-peptide (38 µM and 9 µM, respectively).Indomethacin, a non-steroidal anti-inflammatory drug, also showed an inhibition on LPS-induced MCP-1 production.The inhibitory effect of indomethacin (0.25 mM) on MCP-1 production was found to be similar to that of Chlorella 11-peptide in a low dose (9 µM) but less potent in comparison to a high dose (38 µM) of Chlorella 11-peptide.
Studies have shown that LPS induces production of proinflammatory cytokines (e.g., TNF-α, IL-1, IL-6) which contribute to vascular inflammation and atherosclerosis [19] and the occurrence of atherosclerosis is initiated by adhesion of monocytes to activated endothelial cells [21].In the present study, we found that LPS significantly induced MCP-1 production in macrophages at 12 h (Figure 1).Therefore, the supernatant of LPS-stimulated macrophage cells cultured at 12 h was applied to induce adhesion molecules in SVEC4-10 endothelial cells and these induced molecules in relation to Chlorella 11-peptide can then be evaluated.

Inhibitory Effects of Chlorella 11-Peptide on E-Selectin, ICAM-1 and VCAM-1 Production Induced by 50% RAW-Conditioned Medium
Recombined MCP-1 and other proinflammatory cytokines (recombined TNF-α and IL-6), as mentioned in the introduction of the manuscript, are capable of inducing adhesion molecules.These evidences were all well demonstrated by others' work and have been tested in our preliminary experiments (data not shown).In addition, in our experiments we also found that Chlorella 11-peptide-treated RAW medium was not able to induce adhesion molecules production as a control (data not shown).E-selectin production in SVEC4-10 endothelial cells was significantly induced by 50% RAW conditioned medium (p < 0.005, Figure 2).Both the high (38 µM) and the low (9 µM) concentrations of Chlorella 11-peptide were able to significantly decrease the E-selectin production (p < 0.005 and p < 0.01, respectively).However, indomethacin did not affect the production of E-selectin induced by 50%RAW-conditioned medium.There was about a 5-fold increase in ICAM-1 production when SVEC4-10 endothelial cells were stimulated with 50%RAW-conditioned medium (Figure 3).The increased ICAM-1 production was significantly inhibited by Chlorella 11-peptide and indomethacin (p < 0.005, Figure 3).VCAM-1 production was also induced with 50% RAW-conditioned medium (Figure 4).Notably, neither the low concentration of Chlorella 11-peptide nor indomethacin exhibited any inhibition on VCAM-1 induction.However, the high concentration of Chlorella 11-peptide significantly suppressed the VCAM-1 production (p < 0.005).Normally E-selectin expressed by endothelial cells is low but cytokines, reactive oxygen species, and bacterial endotoxin can elicit its expression [24].ICAM-1 is constitutively expressed on endothelial cells in most regional vascular beds, and its expression can be significantly increased with cytokines or bacterial endotoxin.In comparison with ICAM-1, VCAM-1 predominantly mediates the adhesion of lymphocytes and monocytes upon stimulation [25].Amberger et al. reported a low VCAM-1 gene expression of human umbilical vein endothelial cells after TNF-α stimulation [26].Importantly, all of these elevated adhesion molecules in positive relation to atherosclerosis were substantially reduced by the presence of Chlorella 11-peptide (38 µM).Indomethacin is a potent antiinflammatory agent.However, its inhibitory effects on proinflammatory cytokine-induced adhesion molecules (e.g., E-selectin, VCAM-1) were not as effective as shown in Figures 2 and 4.Moreover, compared to indomethacin (Figure 4), Chlorella 11-peptide can effectively alleviate both the production of ICAM-1 and VCAM-1 which are responsible for leukocyte transmigration (ICAM-1) and leukocyte-endothelium signal transduction (VCAM-1) [27,28].
Cyclic AMP is a ubiquitous regulator of inflammatory and immune reactions.Mediation of cell cross talk through adhesion molecules (e.g., ICAM-1, E-selectin, VCAM-1) has been reported to be dependent on intracellular cAMP [29][30][31].In this study, we used an inhibitor of protein kinase A, H89 [26], to study whether inhibitory effects of Chlorella 11-peptide on ICAM-1 and VCAM-1 production were mediated via the cAMP pathway.We showed that addition of H89 can compromise the inhibitory effects of Chlorella 11-pepetide on the induced ICAM-1 and VCAM-1 production (Figures 3 and 4).This indicates that cAMP pathway was likely involved in the inhibitory actions of the peptide.The counteraction of H89 to indomethacin on the induced VCAM-1 production was not observed (Figure 4).The possible explanation is that Indomethacin requires much higher concentration (above 20 mM) to inhibit cAMP-dependent protein kinase activity [32], which is much higher than the concentration (0.25 mM) used in this study.
ET-1 mRNA expression was reported to be elevated in the artery with atherosclerotic lesion [16].Antagonism of the ET-1 receptors was able to reduce the atherosclerotic lesion formation [33].Thus, blocking ET-1 has been considered to be a strategy for prevention of atherosclerosis [15].In this study, data showed that Chlorella 11-peptide is able to significantly suppress the ET-1 mRNA expression and holds a great potential in anti-atherosclerosis uses.

Inhibitory Effects of Chlorella 11-Peptide on Intercellular Permeability of Endothelia
After stimuli of 50% RAW-conditioned medium, the SVEC4-10 endothelial intercellular permeability showed a great increase (p < 0.005, Figure 6).Both the high and the low doses of Chlorella 11-peptide inhibited the increased intercellular permeability (p < 0.005).Moreover, this inhibition on the intercellular permeability was not observed in the indomethacin-treated group.
Endothelial permeability is controlled in part by the dynamic opening and closing of endothelial cell-cell junctions which relates to the interactions between endothelial cells and the extracellular matrix [34,35].Increased endothelial permeability to lipoproteins or immune cells is considered as an initiating step of atherosclerosis pathogenesis, and the accumulation of debris in the intima would result in atherosclerotic plaques [21].Therefore, maintaining the endothelial permeability unaffected by proinflammatory cells would help to decrease the plaque formation [36].Our permeability assay showed that Chlorella 11-peptide possesses a potent ability to inhibit the increased intercellular permeability of SVEC4-10 endothelia induced by 50% RAW-conditioned medium (Figure 6), which indicates the effectiveness of the peptide in preventing the development of atherosclerosis.

Chlorella-11 Peptide Preparation
The Chlorella-11 peptide of Chlorella pyrenoidosa was prepared from algal protein waste essentially as described previously [19,20].The algal protein waste was obtained from the insoluble material after hot water extraction of Chlorella pyrenoidosa.Then, the algal protein waste (10%, w/v) was digested with commercial proteases (pepsin, flavourzyme, alcalase, and papain) at the concentration of 0.2% (w/v) for 15 h at the designate pH and temperature for each enzyme reaction, using the reaction conditions provided by the manufacturer.At the end of the reaction, the digestion was heated in a boiling water bath for 10 min in order to inactivate the enzyme.The resulted non-soluble material was subsequently spray-dried and was extracted with methanol (1:10 w/v) for three times.After being concentrated, the MeOH-extracted fraction was then extracted by distilled water again prior to freeze-drying.
The isolation and purification of Chlorella 11-peptide was as described previously with modification [19,20].Briefly, ammonium sulfate was added to precipitate proteins and the precipitated proteins were removed by centrifugation (10,000× g, 30 min) and dissolved in a small volume of distilled water.The solution was fractionated using a Sephacryl S-100 high HR column ( 2.6 × 70 cm) to detect peptides at OD 210 nm.The fraction was collected and subsequently loaded onto a Q-sepharose Fast Flow column ( 2.6 × 40 cm), which was pre-equilibrated with a 20 mM Tris-HCl buffer solution (pH 7.8).The separation was performed with 1.0 M NaCl in the same buffer solution and fractions were collected, dialyzed in deionized water, and lyophilized.The peptide concentration was determined with Pierce micro BCA protein assay (Thermo Fisher Scientific Inc., Waltham, MA, USA).The molecular weight of peptides was determined by using a Superdex peptide HR 10/30 column at a flow rate of 0.5 mL/min.The standard curve was established with cytochrome c (MW 12327 Da), approtonin (MW 6500 Da), gastrin (MW 2098 Da) and Leu-Gly (MW 188 Da).
The peptide fraction was further separated successively by a reversed-phase HPLC column, and a purified peptide was attained.The purity and identity of the final preparation was confirmed by mass spectrometric analysis on an Agilent 6510 Q-TOF mass spectrometer (>95%) and Edman degradation (see Supplementary Information).The sequence of Chlorella 11-peptide was determined as Val-Glu-Cys-Tyr-Gly-Pro-Asn-Arg-Pro-Gln-Phe, with a molecular mass of 1309 Da.In later parts of our work, we used the synthetic peptide provided by Kelowna International Scientific Inc. (Taiwan).

MCP-1 Assay
The RAW264.7 cells were seeded at a density of 2 × 10 4 cells/well in 96-well plates.Subsequently, the cells were incubated in the presence of LPS (1 µg/mL) with/without Chlorella 11-peptide (9 µM and 38 µM) for 12 hours prior to the culture medium was collected for MCP-1 measurement.This culture supernatant (without peptide treatment) was also taken to prepare "50% RAW-conditioned medium" (see the next section).The concentration range of Chlorella 11-peptide was chosen based on our previous finding [19].Indomethacin (0.25 mM) was used as a positive control.MCP-1 levels in culture media were determined with commercial ELISA assay kits.

Adhesion Molecules-E-Selectin, ICAM-1 & VCAM-1 Level Measurements in SVEC4-10 Endothelial cells
SVEC4-10 cells (ATCC number: CRL-2181), which are well differentiated, responding like normal endothelial cells to some interleukins and to extracellular matrix signals for tube-like differentiation.SVEC4-10 was demonstrated to retain morphological and functional characteristics of normal EC [37].SVEC4-10 endothelial cells were obtained from Bioresource Collection & Research Center (Taiwan) and cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% Fetal Bovine Serum, 1 mM sodium pyruvate, 100 U/mL penicillin and 100 µg/mL streptomycin.Cells were maintained in 100 mm petri dishes at 37 °C in a humidified atmosphere containing 5% CO 2 .The SVEC4-10 cells were seeded at a density of 2 × 10 4 cells/well in 96-well plates overnight prior to treatments.For stimuli, the culture medium of SVEC4-10 cells was changed to "50% RAW-conditioned medium" (made of equal volume of SVEC4-10 medium and the supernatant of LPS-stimulated RAW264.7 culture medium) with and without Chlorella 11-peptide.Indomethacin was dissolved in 95% ethanol and applied as a positive control and cells were incubated for further 24 h prior to E-selectin and ICAM-1 assays and 6 h prior to VCAM-1 assay.The concentration of these adhesion molecules were measured with commercial ELISA assay kits.

BSA Transwell Permeability Assay
SVEC4-10 cells were seeded (1 × 10 5 cells/insert) on gelatin-coated (1%) polystyrene filters (Costar Transwell, pore size = 0.4 mm, Corning Inc., Taiwan), allowed to grow to confluence on Transwell inserts and then replaced with FBS-free medium for additional 3 h [38].An FBS-free medium containing 10 mg/mL BSA was placed into the upper compartment and an FBS-free medium without BSA was placed in the lower compartment of the Transwell.The transfer rate across the cell monolayer was assessed by measuring the increased amount of BSA in the lower compartment after 30 min.BSA was quantified using Bradford reagent.

Statistical Analysis
Data of results (n ≥ 8) from different experimental days were statistically analyzed for MCP-1, E-selectin, ICAM-1, VCAM-1, and intercellular permeability assays using Prism software (GraphPAD Inc., La Jolla, CA, USA).A two-tailed Student's unpaired test was applied to compare the mean values of two populations of continuous data.The normal distribution and variance of each group was the same.Electrophoresis gel data were performed (n ≥ 3) and a representative example is shown in the results.

Conclusions
The expression of various CAMs by activated endothelial cells is a rate-determining step in recruiting inflammatory cells and plays a pivotal role in the progress of CVD [6,24].Our previous study clearly showed that peptides from Chlorella are a very effective inhibitor of TNF-α and IL-6 production in macrophages [19].In this study, we further demonstrate that Chlorella 11-peptide could not only prevent LPS-induced MCP-1 production in RAW264.7 macrophages, but also effectively inhibit the production of adhesion molecules in endothelial cells.In addition, this peptide exhibited to alleviate ET-1 mRNA expression and maintain endothelial permeability unaffected under the influence of proinflammatory cytokines.By combining these results, Chlorella 11-peptide may become a potential biomolecule in ameliorating the development of atherosclerosis.