Conceived and designed the experiments: JHC SYH KHL IM YIM SHL. Performed the experiments: HJJ DLL KHL SHK YSH. Analyzed the data: HJJ SHL. Contributed reagents/materials/analysis tools: JHC SHL. Wrote the paper: SHL JHC. Revised article critically for important intellectual content: YIM.
The authors have declared that no competing interests exist.
Saturated fatty acids are known to activate macrophages and induce vascular inflammation. Although cytokines from activated macrophage influence other vascular cells, the influence of saturated fatty acids on the paracrine effect of macrophages is not fully understood yet. Here we examined the impact of palmitate on the effect of macrophages on vascular smooth muscle cells (SMCs) and their mediators. SMCs proliferation increased significantly after treatment with conditioned media from palmitate-stimulated RAW264.7 cells. SMC migration was found to be greater after treatment with palmitate-conditioned media. SM α-actin and SM22α were decreased in SMCs treated with palmitate-conditioned media. When stimulated with palmitate, RAW264.7 cells secreted more bone morphogenetic protein (BMP)2 and BMP4 into the cell culture media. SMC proliferation, migration, and phenotypic changes were attenuated after treatment of neutralizing antibodies against BMPs or knockdown of BMPs with siRNA. The influences of these proteins were further confirmed by direct treatment of recombinant BMP2 and BMP4 on SMCs. Particularly, the effects of BMPs on SMC migration on phenotypic change were obvious, whereas their effect on SMC proliferation seemed not significant or modest. In conclusion, palmitate promoted macrophages' paracrine effects on SMC proliferation, migration, and phenotypic change. The effect of stimulated macrophages was mediated, at least in part, by BMP2 and BMP4. These results suggest a novel mechanism linking saturated fatty acids and the progression of vascular diseases that is possibly mediated by BMPs from macrophages.
Free fatty acid levels are often elevated in obese individuals and patients with metabolic syndrome or diabetes, and predicts cardiovascular events
The proliferation and phenotypic changes of vascular smooth muscle cells (SMCs) from a quiescent and contractile to a synthetic form are critical in atherosclerosis. SMCs interact with other vascular cells including endothelial cells, monocytes, and macrophages and these interactions can influence SMC phenotypes. Known factors involved in the modulation of SMC phenotypes include growth factors such as platelet-derived growth factor (PDGF), angiotensin II, interleukins, and mechanical stimulation
Bone morphogenetic proteins (BMPs) constitute a large group in the transforming growth factor-β superfamily
Here, we examined the impact of palmitate on the paracrine effects of macrophages on vascular SMCs. We investigated the effects of palmitate-stimulated macrophages on SMC proliferation, migration, and phenotypic change. We hypothesized that BMPs could mediate macrophage-dependent SMC changes and demonstrated the role of BMP2 and 4 in the interactions between these two cell types.
Sodium salt of palmitate, bovine serum albumin (BSA; fatty acid-free and low endotoxin), phorbol 12-myristate 13-acetate (PMA), β-mercaptoethanol, mouse monoclonal antibodies against smooth muscle α-actin (SM α-actin) and SM22α were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's modified Eagle's medum (DMEM), RPMI 1640 medium, gentamycin, fetal bovine serum (FBS) and Dulbecco' phosphate buffered saline (PBS) with Ca2+ and Mg2+ were obtained from Gibco (Grand Island, NY, USA). Neutralizing antibodies against BMP2 and BMP4 were obtained from LSBio (Seattle, WA, USA) and Abcam (Cambridge, MA, USA), respectively. Isotype-matched control IgG and recombinant BMPs were purchased from R&D Systems (Minneapolis, MN, USA).
Palmitate was dissolved in 0.1 M NaOH/70% ethanol at 70°C. It was then complexed with 10% fatty acid-free low endotoxin BSA at 55°C for 10 minutes. A stock solution of 50 mM palmitate was prepared before the experiment. Palmitate was used at a concentration of 250 µM and the solution was adjusted to a pH of 7.4. Palmitate preparation was assessed for lipopolysaccharide contamination with Limulus Amebocyte Lysate Assay (Lonza, Basel, Switzerland), and the endotoxin level was <0.05 EU/mL through all experiments. Control solution containing ethanol and BSA was prepared similarly.
Rat aortic smooth muscle cells (SMCs) were obtained from BioBud Inc. (Seoul, Korea) and grown in DMEM containing 10% heat-inactivated FBS, 100 U/mL penicillin and 100 µg/mL streptomycin. RAW264.7 cells and human monocyte leukemia cell line THP-1 were purchased from the Korean Cell Line Bank (Seoul, Korea). RAW264.7 cells were grown in DMEM with 10% FBS. THP-1 cells were maintained in medium supplemented with 10% FBS and 0.2% β-mercaptoethanol.
SMC proliferation was examined using a BrdU cell proliferation assay kit (Millipore Chemicon, Billerica, MA, USA) according to the manufacturer's protocol.RAW264.7 cells were stimulated with BSA control or palmitate (250 µM)for 4 hours. Conditioned media were collected 20 hours later. PDGF that is known to induce SMC proliferation
SMCs were gently scraped with pipette tips. They were then serum-starved for 24 hours and treated with conditioned media from macrophages that had been stimulated with BSA control, palmitate (250 µM) or they were treated with PDGF (25 ng/mL)/IL-1β (10 ng/mL). Twenty-four hours later, the wound sites along the scratches were examined and photographed at 100-fold magnification. Widths of wound gaps after 24 hours were measured on the photographs for comparison. Boyden chamber assay was conducted using 8.0-µm pore transwell inserts (Corning, Corning, NY, USA). Briefly, serum-starved SMCs were seeded in the upper chamber (250 µL, 1×105 cells/well in a 24-well plate). Cell migration was stimulated by 750 µL of conditioned media or PDGF/IL-1β to the lower well of the Boyden chamber. After 4-24 hours of incubation, the surface of the upper membrane was swabbed with a cotton-tipped applicator to remove non-migrating cells. Inserts were fixed in methanol for 30 minutes and stained with 1% crystal violet for 2 hours. For quantitative analysis, the surface of membrane was eluted by methnol and optical density was measured using a microplate reader. Boyden chamber assay to test the effect of recombinant BMPs was performed with the same manner.
The effect of palmitate-stimulated macrophages on SMC phenotype was assessed by immunoblotting. Total cell lysates were collected from the cells. Cell extracts were subjected to 10% SDS-PAGE and proteins were electrotransferred to a polyvinylidene difluoride membrane for immunoblot analyses. The membrane was blocked in phosphate-buffered saline containing 0.05% Tween 20 and 5% nonfat dry milk. Immunoblotting was performed with anti-SM α-actin. The reactive bands were visualized with Supersignal West Dura Extended Duration Substrate (Thermo Scientific, Waltham, MA, USA). In the experiments using conditioned media, RAW264.7 cells were treated with BSA control or palmitate (250 µM) for 4 hours and the conditioned media were collected 20 hours later. SMCs were serum-starved for 24 hours and treated with the conditioned media or PDGF (25 ng/mL)/IL-1β (10 ng/mL) for 24 hours. Steps of immunoblotting thereafter were the same as described above. Primary antibodies were: anti-β-actin, anti-SM α-actin, and anti-SM22α. Immunoblotting to evaluate the effect of recombinant BMPs on SMC phenotypic change was performed with the same manner. BMP proteins were detected by immunoblotting in conditioned media after pull-down using heparin Sepharose beads.
Total RNA from cells was isolated using the RNA extraction reagent Trizol (Invitrogen, Carlsbad, CA, USA). The cDNA was synthesized using Superscript III reverse transcriptase (Invitrogen) according to manufacturer's protocol. Real-time PCR was performed to determine the mRNA levels of BMP2 and BMP4 in cells using LightCycler FastStart DNA Master SYBR Green I mix (Roche Applied Science, Indianapolis, IN, USA) with a LightCycler 480 System (Roche Applied Science), in accordance with the manufacturer's instructions. The primers used were as follows: BMP2,
To neutralize the effects of BMPs, we used commercially available antibodies against human BMP2 and BMP4. THP-1 cells were differentiated into macrophages using 100 nM PMA in media with 10% FBS. Three days later, the cells were washed with PBS and incubated for another 22 hours in the same medium but without FBS. On the day of the experiment, the cells were incubated for 4 hours with 250 µM palmitate or BSA control. Conditioned media were collected 20 hours later. Conditioned media were treated with 2 µg/mL of anti-BMP2, anti-BMP4 antibodies, or both for 1 hour. Antibody-treated conditioned media were added to serum-starved SMC for 24 hours.
BMP2 siRNA, BMP4 siRNA and scrambled control siRNA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). THP-1 cells differentiated with PMA were transfected with 10 nM of siRNA using Lipofectamine RNAiMAX Reagent (Invitrogen) according to the manufacturer's protocol. After 24 hour of siRNA transfection, we determined BMP2 and BMP4 levels in whole cell lysates by immunoblot analysis to confirm the silencing of these proteins. The cells transfected with BMP siRNA were also incubated for 4 hours with 250 µM palmitate-BSA complex or BSA control and conditioned media were collected 20 hours later.
All data are presented as the mean ± standard error of the mean (SE). Statistical analysis between two groups was conducted using Student's
The effects of palmitate-stimulated macrophages on SMC proliferation were evaluated by BrdU assay. Proliferation of SMCs increased 48 hours after treatment with conditioned media from RAW264.7 cells that had been stimulated with palmitate. This effect was modest compared to that of PDGF/IL-1β. The mean increase was 26–31% for palmitate and 94–132% for PDGF/IL-1β at 48–72 hours (
A. To evaluate SMC proliferation, RAW 264.7 cells were stimulated with BSA control, palmitate (250 µM) for 4 hours. Conditioned media were collected 20 hours later. SMCs were serum-starved for 24 hours and treated with each conditioned media or PDGF (25 ng/mL)/IL-1β (10 ng/mL). BrdU incorporation assay showed that palmitate-conditioned media modestly increased cell proliferation compared to the control. B. In the wound healing assay, SMCs were gently scraped with a pipette tips. SMCs were serum-starved and treated with each conditioned media or PDGF/IL-1β as described in BrdU assay. Wound sites along the scratches were examined and photographed at 100-fold maginification. C. The width of the wound gap was measured on the photograph and expressed as % of control. The wound gaps were shorter in cells treated with palmitate-conditioned media or PDGF/IL-1β. D. In Boyden chamber assay, SMCs were incubated with each conditioned media or PDGF/IL-1β in the chamber for 4-24 hours. After crystal violet staining, migrated cells in the filters were observed under a microscope (Olympus, Tokyo, Japan). E. In addition, after crystal violet staining, the surface of membrane was eluted by methanol and optical density was measured using a microplate reader. Treatment with palmitate-conditioned media or PDGF/IL-1β induced significant SMC migration. Data are means ± SE of three independent experiments. *p<0.05 compared to treatment with conditioned media from BSA control.
The effects of palmitate-stimulated macrophages on SMC migration were assessed by a wound healing and Boyden chamber assay. In the wound healing assay, the widths of wound gaps were shorter in cells treated with palmitate-conditioned media or PDGF/IL-1β (
To examine the paracrine effects of palmitate-stimulated macrophages on SMC phenotype, we determined the effects of conditioned media from palmitate-stimulated macrophages on SMC contractile markers. RAW264.7 cells were stimulated with BSA control or palmitate. SMCs treated with the conditioned media from palmitate-stimulated cells or PDGF/IL-1β showed a decrease in SM α-actin and SM22α compared to those treated with the conditioned media from BSA control-treated cells (
A. RAW 264.7 cells were treated with BSA control orpalmitate for 4 hours and conditioned media were collected 20 hours later. SMCs were serum-starved for 24 hours and treated with conditioned media or PDGF/IL-1β for 24 hours. Immunoblots showed decreased SM α-actin and SM22α in cells treated with conditioned media from palmitate-stimulated macrophages. B. The band intensities were determined by quantitative densitometry. Data are illustrated in arbitrary integrator units relative to β-actin and represent the mean ± SE from three independent experiments. *p<0.05 compared to treatment with conditioned media from BSA control.
We examined conditioned media from palmitate-stimulated macrophages to evaluate the mechanism of the effects on SMC. When stimulated with palmitate, RAW264.7 cells secreted BMP2 and BMP4 into cell culture media (
A. RAW 264.7 cells were treated with BSA control or palmitate (250 µM) for 4 hours and conditioned media were collected 20 hours later. BMP 2 and 4 secreted in the media were pulled down on heparin-Sepharose columns and detected by immunoblotting. B. RAW 264.7 cells were treated with BSA control or palmitate for 4 hours, and then incubated with fresh media for 20 hours. BMP2 and BMP4 mRNA levels were analyzed by real-time PCR. Data are illustrated in arbitrary unit relative control and represent the means ± SE from three independent experiments. *p<0.05 compared to control.
A. THP-1 cells differentiated with PMA were stimulated with BSA control or palmitate for 4 hours and conditioned media were collected 20 hours later. Palmitate-conditioned media were treated without or with 2 µg/mL of anti-BMP2, anti-BMP4, or both for 1 hour. Each conditioned media was added to serum starved SMCs for 72 hours. Cell proliferation was analyzed by BrdU incorporation assay. Promotion of SMC proliferation by palmitate-conditioned media was abrogated by combined treatment of anti-BMP2/anti-BMP4. B. Each conditioned media described above was added to SMCs for 24 hours. Cell migration assay was conducted using Boyden chamber assay. Promotion of SMC migration by palmitate-conditioned media was inhibited by treatment of anti-BMP2 or anti-BMP4 or combination of both. C. Each conditioned media described above was added to SMCs for 24 hours. Immunoblots showed that anti-BMP4 or anti-BMP2/anti-BMP4 combination inhibited phenotypic change of SMCs. D. The band intensities were determined by quantitative densitometry. E. THP-1 cell-derived macrophages were transfected with BMP2 or BMP4 or both siRNA for 24 hours. Immunoblots demonstrated knockdown of BMP2 or BMP4 or both BMPs in the macrophages. F. SMCs were treated with palmitate-conditioned media from macrophages without or with knockdown of BMPs for 24 hours. Equal amount of protein were separated by SDS-PAGE, and immunoblots showed that knockdown of BMP4 or both BMPs abrogated phenotypic change of SMCs. Data are illustrated in arbitrary integrator unit relative to β-actin and represent the means ± SE from three independent experiments. *p<0.05 compared to control. **p<0.05 compared to palmitate-conditioned media treatment.
A. Quiescent SMCs were treated with various concentration of BMP2 or BMP4 for 24 hours. Cell proliferation was analyzed by BrdU incorporation assay. Recombinant BMP2 and BMP4 did not have obvious effects on SMC proliferation. B. SMC migration assessment was conducted using Boyden chamber assay and BMP2 and BMP4 significantly promoted SMC migration. C. SMCs were treated with various concentration of recombinant BMP2 or BMP4 for 24 hours. Equal amount of protein was separated by SDS-PAGE and immunoblots showed both recombinant BMPs induced phenotypic change of SMCs with lowering expression of SM α-actin and SM22α.
This study demonstrated that palmitate influences the paracrine effects of macrophages. Palmitate-stimulated macrophages promoted SMC proliferation and migration. In addition, these macrophages decreased the levels of SMC contractile markers, indicating that SMCs were changed into more synthetic forms. These effects of palmitate-stimulated macrophages required the secretion of BMP2 and BMP4, as demonstrated by the attenuation of SMC proliferation, migration, and phenotypic changes after treatment with neutralizing antibodies against BMPs or knockdown of BMPs with siRNA. The influences of these proteins were further confirmed by direct treatment of recombinant BMP2 and BMP4 on SMCs. The effect of BMP4 was more consistent compared to that of BMP2. Particularly, the effects of BMPs on SMC migration or phenotypic change were more obvious, whereas their effect on SMC proliferation seemed not significant or modest at maximum. Collectively, these findings suggest a new mechanism linking elevated saturated fatty acids and the progression of vascular diseases, possibly mediated by BMP2 and BMP4 originating from macrophages.
Prior studies have provided evidence for a proinflammatory effect of saturated fatty acids, particularly palmitate. In mouse and human cells, palmitate induces cyclooxygenase-2
We found that BMP4 played a role in changing SMC phenotype to the synthetic form. Several groups of investigators have documented the effect of BMP4 in vascular cells. Sorescu et al. reported that BMP4 produced in endothelial cells stimulates monocyte adhesion which is dependent on NFκB and intercellular adhesion molecule 1
BMP2 expression was found to be upregulated in atherosclerotic lesions
We note that our study has some limitations. First, a variety of inflammatory mediators including growth factors, interleukins, and TNF-α are involved in SMC phenotypic change, growth, and migration
In conclusion, palmitate-stimulated macrophages promoted vascular SMC proliferation and migration, and changed SMCs to a more synthetic form. The effects of these stimulated macrophages on SMC migration and phenotypic change were mediated, at least in part, by BMP2 and BMP4. Our results suggest a novel mechanism linking elevated saturated fatty acid levels and the progression of vascular diseases, possibly mediated by BMPs produced by macrophages.