Effect of serum concentration on adhesion of monocytic THP-1 cells onto cultured EC monolayer and EC-SMC co-culture

Effect of serum concentration on adhesion of monocytic THP-1 cells onto cultured EC monolayer and EC-SMC co-culture 1 2 first-author 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:karino@es.hokudai.ac.jp †E-mail:karino@es.hokudai.ac.jp karino@es.hokudai.ac.jp Laboratory of Biofluid Dynamics, Research Institute for Electronic Science, Hokkaido University, Sapporo 060-0812, Japan Laboratory of Biofluid Dynamics, Research Institute for Electronic Science, Hokkaido University, Sapporo 060-0812, Japan Department of Stomatology, School of Medicine, Zhejiang University, Hangzhou 310006, China Department of Stomatology, School of Medicine, Zhejiang University, Hangzhou 310006, China Background: 2 The adhesion of monocytes to the endothelium following accumulation of low-density lipoprotein (LDL) in subendothelial spaces is an important step in the development of intimal hyperplasia in arterially implanted vein grafts and atherosclerosis in both animals and humans. However, it is not well known how serum factors affect the adhesion of monocytes. Methods: 2 We have studied the effect of fetal calf serum (FCS), which we considered a source of LDL, on the adhesion of monocytes to endothelial cells (ECs) by using human monocytic THP-1 cells and both a monolayer of cultured bovine aortic endothelial cells (EC monoculture) and a co-culture with bovine aortic smooth muscle cells (EC-SMC co-culture). Results: 2 It was found that the addition of FCS to the medium greatly affected the adhesion of THP-1 cells, and the higher the concentration of FCS in the medium, the greater the adhesion of THP-1 cells to endothelial cells. Adhesion of THP-1 cells to an EC-SMC co-culture was approximately twofold greater than that to an EC monoculture, and after adhering to endothelial cells, many THP-1 cells transmigrated into the layer of smooth muscle cells. Conclusion: 2 The results suggest that the elevation of the LDL (cholesterol) level in blood provides a favorable condition for the development of intimal hyperplasia and atherosclerosis by promoting the adhesion of monocytes to the endothelium and their subsequent migration into subendothelial spaces. Endothelial cells (ECs) Smooth muscle cells (SMCs) Monocyte THP-1 cells Low-density lipoprotein (LDL) Adhesion Transendothelial migration Serum concentration INTRODUCTION 1 Adhesion of monocytes to the endothelium and their migration into subendothelial spaces are important steps in the development of intimal hyperplasia in arterially implanted vein grafts and atherosclerosis in both animals and humans (Gerrity, 1981a 1981a ; 1981b 1981b ; Zwolak et al., 1989 1989 ; Michison et al., 1990 1990 ; Itoh et al., 1994 1994 ; Stark et al., 1997 1997 ). It occurs preferentially in regions where low-density lipoproteins (LDLs, main carrier of cholesterol in blood) and oxidized low-density lipoproteins (oxLDLs) are accumulated in subendothelial spaces (Schwenke and Carew, 1989 1989 ; Back et al., 1995 1995 ; Malinauskas et al., 1995 1995 ; Steinberg and Lewis, 1997 1997 ). Since intimal hyperplasia and atherosclerosis tend to occur under the condition of hypercholesterolemia both in animals and humans, it is considered that the adhesion of monocytes to the endothelium and their subsequent migration into subendothelial spaces are affected by the concentrations of LDL and adhesive proteins in blood. In connection with this, Kinard et al.( 2001 2001 ) studied the effects of serum factors on adhesion and transendothelial migration of porcine monocytes and THP-1 cells to a porcine endothelial cell (EC) monolayer and co-culture with smooth muscle cells (SMCs). They showed that the adhesion of THP-1 cells to ECs incubated in serum-containing and serum-free media did not differ significantly. However, they did not test the dose effect of the serum. Therefore we have studied the dose effect of fetal calf serum (FCS), which we considered a source of LDL, on the adhesion of monocytes to ECs by using human monocytic THP-1 cells and both a monolayer of cultured bovine aortic endothelial cells (EC monoculture) and a co-culture with smooth muscle cells (EC-SMC co-culture). MATERIALS AND METHODS 1 Materials 2 Bovine aortic ECs and SMCs were purchased from Cell Systems Inc. (Kirkland, WA, USA) and Cell Applications Inc. (San Diego, CA, USA), respectively. Human monocytic leukaemia cell line THP-1 cells were obtained from a repository reference seed stock (depositor: Dr. J. Clarke, AVRI, Pirbright, UK). Calcein-AM used as a fluorescent dye was purchased from Trevigen, Inc. (Gaithersburg, MD, USA). FCS was purchased from Trace Scientific Co., Ltd. (Melbourne, Australia). L-ascorbic acid, fibronectin, penicillin, streptomycin and Iscov’s modified Dulbecco’s medium (IMDM) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Preparation of EC monocultures and EC-SMC co-cultures 2 At first, bovine aortic SMCs and ECs were cultivated separately in culture dishes with IMDM containing 10% (v/v) FCS, 100 IU/ml penicillin and 100 µg/ml streptomycin at 37 °C in 5% (v/v) CO 2 and 95% (v/v) air humidified atmosphere, and cells at Passages 5~11 were used to prepare EC monocultures and EC-SMC co-cultures. After obtaining a sufficient number of SMCs, the cells were released from the cell culture dish with a trypsin-EDTA (ethylene diamine tetraacetic acid) solution, centrifuged, and then suspended in IMDM containing 20% (v/v) FCS, 100 IU/ml penicillin, 100 µg/ml streptomycin and 50 µg/ml L-ascorbic acid. To prepare an EC-SMC co-culture used as a model of an arterial wall, the SMCs were firstly seeded onto 24-well culture plates at 5.0×10 5 cells/cm 2 and cultivated for 3 d in 2 ml of culture medium, and then ECs were seeded at 5.0×10 5 cells/cm 2 directly over the SMCs. Then they were co-cultivated for 3 d in the same culture medium until the ECs became confluent and completely covered the SMCs, as shown in Fig. 1 1 (see Page 627), by the uptake of DiI-labeled acetylated LDL (Ac-LDL), forming an EC-SMC co-culture system. At the same time, monocultures of ECs were also prepared using the same method and cultivated for 3 d until the cells became confluent. Cultivation of THP-1 cells 2 Human monocytic leukaemia cell line THP-1 cells were cultivated in suspension in IMDM containing 10% FCS, 100 IU/ml penicillin and 100 µg/ml streptomycin at 37 °C in 5% CO 2 and 95% air humidified atmosphere. Preparation of fluorescent THP-1 cells 2 To visualize the THP-1 cells adherent to the EC monoculture and the EC-SMC co-culture, the THP-1 cells were labeled with a fluorescent dye, calcein-AM, by incubating 2.0×10 6 cells in 4 ml of IMDM containing 10% FCS and 40 µl of calcein-AM to make a final calcein-AM concentration of 10 µmol/L at 37 °C for 1 h. Dye loading was stopped by adding 4 ml of cold IMDM containing 10% FCS to the suspension and then centrifuging. Fluorescence-labeled cells were resuspended in 4 ml of IMDM containing 10% FCS and prepared for use in adhesion experiments. Procedures for adhesion experiments of THP-1 cells 2 To investigate the effect of the concentration of serum added to a cell culture medium as a source of LDLs on the adhesion of THP-1 cells to the endothelium, both EC monocultures and EC-SMC co-cultures were divided into 3 groups and the media were discarded. The cells in the EC monoculture and EC-SMC co-culture were rinsed 3 times with 1 ml of phosphate-buffered saline (PBS, pH 7.3) to wash out the FCS. Then 4 ml of new culture medium containing antibiotics and FCS either at a concentration (v/v) of 20%, 1% or 0.1% was put in each dish and they were incubated overnight at 37 °C in 5% CO 2 and 95% air humidified atmosphere to get ready for use in adhesion experiments of THP-1 cells. The EC monocultures and EC-SMC co-cultures were taken out of the incubator. Then 20~30 µl of suspension of calcein-AM-labeled THP-1 cells containing 1.0×10 4 cells was added to each culture dish and they were incubated for a period of either 30 min, 1 h, 2 h or 4 h at 37 °C in 5% CO 2 and 95% air humidified atmosphere. After that, the suspension of THP-1 cells in each dish was discarded, and the monoculture and co-culture were gently washed three times with 1 ml of PBS at pH 7.3 in order to remove non-adherent THP-1 cells. The number of adherent THP-1 cells was determined by measuring the fluorescence intensity of the adherent THP-1 cells using a Fluoroskan Asent® FL (Thermo Labsystem, Helsinki, Finland) and also by counting the number of adherent fluorescence-labeled THP-1 cells that were seen under a fluorescence microscope (Nikon, Japan) which encompassed a surface area of 0.314 mm 2 . Measurements were carried out at 6 different areas and the results were expressed as the number of THP-1 cells per mm 2 . Observation of THP-1 cells transendothelially migrated into the SMC layer 2 To visualize the THP-1 cells transendothelially migrated into the SMC layer, a special EC-SMC co-culture was prepared by seeding SMCs on the inner surface of an expanded PTFE (polytetrafluoroethylene) graft at 2.0×10 6 cells/cm 2 by means of pressure infusion of a suspension of SMCs using a syringe. The PTFE graft, 3.0 mm i.d. (inner diameter) and 1.0 cm long, was treated with 70% (v/v) ethanol and coated with 0.01% (w/v) fibronectin, an adhesive molecule. The cells were cultivated for 7 d in 40 ml of IMDM containing 20% FCS, 100 IU/ml penicillin, 100 µg/ml streptomycin and 50 µg/ml L-ascorbic acid at 37 °C in 95% air and 5% CO 2 humidified atmosphere. Then, ECs were seeded directly over the SMCs in the same manner as that of SMCs, and they were co-cultivated for 7 d in 40 ml of the same culture medium until the ECs became confluent and completely covered the SMCs, forming an EC-SMC co-cultivated hybrid vascular graft. Then 20~30 µl of a suspension of THP-1 cells containing 1.0×10 4 cells was added to the culture dish containing the hybrid graft and incubated for 4 h at 37 °C in 5% CO 2 and 95% air humidified atmosphere. After that, the suspension of THP-1 cells in the dish was discarded, and the hybrid graft was gently washed three times with PBS at pH 7.3 in order to remove non-adherent THP-1 cells. The cells of the hybrid graft were fixed with a 4% (w/v) formaldehyde solution and embedded in paraffin. Then the graft inbedded in paraffin was sliced into 5-µm thick sections, stained with a hematoxylin-eosin (HE) stain, and observed under a light microscope. Statistical analysis 2 The result was expressed as a mean± SD . The significance in the difference between the means of any two groups was assessed by Student’s t -test. In experiments involving multiple groups, P -values between the means of any two groups were calculated by the analysis of variance (ANOVA) using Scheffe’s test. Differences were considered statistically significant if P < 0.05. RESULTS 1 Effect of serum concentration on adhesion of THP-1 cells 2 Fig. 2 2 (see Page 627) shows the photographs of calcein-AM labeled THP-1 cells adhered to ECs cultivated in media containing serum at 3 different volume concentrations (0.1%, 1% and 20%) after incubation for 1 h. As is evident from Fig. 2 2 , the adhesion of THP-1 cells was affected by the concentration of serum in the medium, and it was the greatest with the cells incubated with a medium containing serum at 20%, among the three concentrations tested. It was also found that the adhesion of THP-1 cells was affected by the duration of incubation as shown in Figs. 3a and 3b 3a and 3b respectively, in terms of fluorescence intensity and the number of THP-1 cells. As shown in Figs. 2 2 and 3 3 , both fluorescence intensity and the number of THP-1 cells that appeared to be adherent to the surface of ECs in the monoculture were the highest at a serum concentration of 20% at any time, and they appeared to decrease over time. Fig. 4 4 shows the results obtained with EC-SMC co-cultures. The tendency of THP-1 cell adhesion in EC-SMC co-culture was almost the same as that in the case of EC monocultures, the highest appearing at a serum concentration of 20% at any time. However, the number of THP-1 cells actually adherent to the surface of endothelial cells was approximately two times higher than that found in the EC monocultures. Effect of the presence of smooth muscle cells on adhesion of THP-1 cells 2 To investigate the effect of the presence of SMCs under the ECs in EC-SMC co-cultures on the adhesion of THP-1 cells to the surface of ECs, we counted the number of adherent THP-1 cells in both the EC monoculture and EC-SMC co-culture after 1-hour incubation with THP-1 cells in a culture medium containing serum at three different concentrations. Fig. 5 5 summarizes the results. As evident from the figure, the adhesion of THP-1 cells was greatly enhanced by the presence of SMCs under a monolayer of ECs. At any serum concentration, the number of adherent cells in the EC-SMC co-culture was approximately twofold of that in the EC monoculture and the difference was statistically significant. Transendothelial migration of THP-1 cells 2 To find out whether the migration of THP-1 cells into subendothelial spaces occurs upon adhesion to ECs, we prepared an EC-SMC co-culture on an expanded PTFE artificial graft, and after finishing adhesion experiments, we made histological specimens of the hybrid graft and observed them under the microscope. Fig. 6 6 shows the crosssection of the hybrid graft obtained after incubation with THP-1 cells for 1 h. It was found that after adhering to ECs, many THP-1 cells migrated across the monolayer of ECs and accumulated in subendothelial spaces. DISCUSSION 1 It is known that under normal physiologic conditions, monocytes do not easily adhere to the endothelium. Adhesion of monocytes occurs following accumulation of LDLs in subendothelial spaces. Since LDLs are present in serum, we considered that the adhesion of THP-1 cells is affected by the concentration of serum in a medium. Therefore in our present study, we tested three different FCS concentrations and obtained a result very different from that of Kinard et al.( 2001 2001 ). Adhesion of THP-1 cells to both the EC monoculture and EC-SMC co-culture increased with an increase in the concentration of serum in the medium. A difference was also seen in the time course of adhesion. Kinard et al.( 2001 2001 ) showed that the adhesion of THP-1 cells increased over time and reached a constant value after 6 h, whereas our results showed a maximum after 2 h and then a gradual decrease over time. Kinard et al.( 2001 2001 ) also tested the adhesion of THP-1 cells to ECs in monoculture or in co-culture with SMCs preincubated in the presence or absence of native LDL, oxLDL or LPC (lysophosphatidylcholine, a component of oxLDL). They found that LPC in a monoculture and LPC and oxLDL in a co-culture enhanced the adhesion of THP-1 cells. Berliner et al.( 1990 1990 ) also showed that oxLDL enhanced the adhesion of monocytes to cultured rabbit aortic ECs by the action of LCP. In their additional experiments, Kinard et al.( 2001 2001 ) found that monocyte chemoattractant protein-1 (MCP-1) produced by SMCs is implicated in both enhanced adhesion and transendothelial migration of THP-1 cells in the same manner as LPC. Cushing et al.( 1990 1990 ) demonstrated that minimally oxidized LDLs, but not native LDLs, induced the production of MCP-1 in vascular wall cells such as ECs and SMCs. In our present study, the number of THP-1 cells adherent to ECs increased with an increase in the concentration of FCS in the medium in both monoculture and co-culture with SMCs. If it is true that native LDLs do not affect the adhesion of THP-1 cells as shown by Kinard et al.( 2001 2001 ), it is likely that oxLDLs modified from native LDLs after being taken up by the ECs during preincubation are responsible for this phenomenon. The decrease in the number of the adherent THP-1 cells that occurred beyond 2-h incubation might be caused either by the decrease in the amount of LDLs in the medium due to the uptake of LDLs and oxLDLs by non-adherent THP-cells or by the disappearance of adhesive molecules. In our present study, we did not try experiments with a serum-free medium since Cai et al.( 2004 2004 ) showed that an insufficient supply of serum causes apoptosis of THP-1 cells. We also found that the adhesion of THP-1 cells was greatly enhanced by the presence of SMCs. The number of THP-1 cells adherent to the ECs of an EC-SMC co-culture was approximately twofold of that of the EC monoculture. Our results are quite the same as those obtained by Kinard et al.( 2001 2001 ) in the presence of oxLDL. In our experiments, it was observed that the number of THP-1 cells adherent to the ECs of the EC-SMC co-culture decreased greatly over time, although the intensity of fluorescence emitted by calcein-AM-labeled THP-1 cells adhering to and presumably taken up in part by the EC-SMC co-culture did not decrease so much. Therefore we prepared histological specimens of the co-culture and examined them under a light microscope. It was found that there were many THP-1 cells in the subendothelial cell layer composed of SMCs, suggesting that most of the THP-1 cells migrated into the subendothelial SMC layer upon their adhesion to ECs. It is likely that LDLs in the serum changed to oxLDL after being taken up by the ECs. Then they were transported to the SMC layer underlying the ECs, and induced the migration of THP-1 cells. 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Atherosclerosis in rabbit vein Grafts The first author (Li-jie Fan) is a Ph.D. investigator invited from Zhejiang University, China by a fund provided by the 21st Century Center of Excellent Program of Hokkaido University, “Advanced Life Science on the Base of Bioscience and Nanotechnology”. The authors thank Prof. Shifang Zhao, Dean of Department of Stomatology, School of Medicine, Zhejiang University, China, for his support and encouragement in our research. 10.1631/jzus.B0820046 Project supported by a Grant-in-Aid for Scientific Research on Priority Areas (No. 15086201) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the Health Bureau of Zhejiang Province (No. 2007B132), China 2008-02-16 2008-06-21 2022-01-05 Copyright © 2008, Journal of Zhejiang University Science 2008 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 08 09