Articles in PresS. Am J Physiol Heart Circ Physiol (November 7, 2014). doi:10.1152/ajpheart.00503.2014
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Akt/eNOS Signaling Pathway Mediates Inhibition of Endothelial
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Progenitor Cells by Palmitate-induced Ceramide
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Minghuan Fu a,1, Zhihong Lib , Tao Tanc, Weixin Guod , Nanzi Xiee, Qing Liua, Hua
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Zhu c* ,Xiaoyun Xiee*, Han Lei a*
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a
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Yuanjiagang District, Chongqing 400016, P.R. China
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b
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423000, China
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c
The First Affiliated Hospital, Chongqing Medical University, No. 1 Youyi Road,
Division of General Surgery, Chenzhou First People′s Hospital, Chenzhou, Hunan
Department of Surgery, Davis Heart and Lung Research Institute, The Ohio State
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University Wexner Medical Center, Columbus, Ohio 43210, the United States
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d
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of Medical sciences No. 106, Zhongshan Road, Guangzhou 510100, China
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e
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Shanghai 200065, China
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Running Title: Ceramide mediates Palmitate-induced EPCs dysfunction
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*Correspondence to:
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Dr. Han Lei
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The First Affiliated Hospital, Chongqing Medical University, No. 1 Youyi Road,
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Yuanjiagang District, Chongqing 400016, P.R. China
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Email: [email protected]
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or Drs. Xiaoyun Xie, [email protected] and Hua Zhu, [email protected]
Guangdong Geriatrics Institute, Guangdong General Hospital, Guangdong Academy
Division of Geriatrics, Tongji Hospital, Tongji University, School of Medicine,
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Copyright © 2014 by the American Physiological Society.
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Abstract
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Palmitate (PA) impairs endothelial progenitor cells (EPCs). However, the molecular
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mechanism underlying the suppressive function of PA remains largely unknown.
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Ceramide, a free fatty acid (FFAs) metabolite, mediates multiple cellular signals. We
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hypothesized that ceramide acts as an intermediate molecule to mediate inhibition of
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EPSc by PA. We first demonstrated that PA could inhibit attachment, migration and
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tube formation of EPCs through suppression of Akt/eNOS signal pathway. In addition,
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we observed PA could induce ceramide accumulation in EPCs. To test whether the
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accumulation of ceramide causes EPCs dysfunction, ceramide synthesis inhibitors,
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myriocin and fumonisin B1 (FB1) were used. We found both inhibitors could
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effectively abolish PA-mediated EPCs inhibition. Furthermore, a ceramide
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deacylation inhibitor, N-oleoylethanolamine (NOE), could augment the inhibitory
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effect of PA on EPCs, indicating it is the ceramide, not its metabolites, that mediates
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suppression of EPCs by PA. We previously showed that Akt/eNOS phosphorylation
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was reduced after PA treatment, which in turn hampered normal bioavailability of
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nitric oxide (NO) leading to impaired functions of EPCs. In order to test the role for
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ceramide in this process, a clinically used NO donor, sodium nitroprusside (SNP), was
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used. We found SNP could rescue the suppressive effects of ceramide on EPCs,
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suggesting ceramide-mediated EPCs inhibition might be through reduction of NO
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production. Taken together, our findings indicated that ceramide induced reduction of
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NO might be the molecular mechanism for PA-mediated EPCs inhibition, thus,
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targeting either ceramide or NO production might be an effective means for
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improvement of EPCs functions in diseases.
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Key words:
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Palmitate, Ceramide, Endothelial Progenitor Cells, Stem Cells, Nitric Oxide
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Introduction
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Endothelial progenitor cells (EPCs), were initially described and defined as a special
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type of stem cell by Asahara et al(2). EPCs have been found in bone marrow, spleen,
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umbilical cord blood (CB) and peripheral blood (PB), and can contribute to the
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formation of new blood vessels in postnatal life and incorporate into injured vessels to
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form mature endothelial cells (ECs) in response to tissue ischemia(9, 28). Increasing
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evidences indicate that incorporation of EPCs play an important role in postnatal
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neovascularization (1, 12).
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In some pathological conditions such as diabetes and coronary artery disease, the
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number of circulating EPCs is decreased and their functions are impaired (8, 22). As
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previously highlighted, circulating EPCs in diabetes are subjected to many
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biochemical alterations attributable to the unfavorable vascular environment. Among
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these factors, elevated levels of free fatty acids(FFAs) are commonly observed in
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human patients with diabetes, obesity, and dyslipidemias. High FFA level is an
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independent risk factor for atherosclerosis, hypertension, and sudden cardiac death(5).
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FFAs may create a state of continuous and progressive damage to the circulating cells.
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Therefore, FFAs may be one of the factors that influences growth and bioactivity of
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EPCs. In deed, it has been reported that the ability of diabetic EPCs to integrate into
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vascular networks was reduced after FFAs treatment(26). In addition, circulating
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EPCs and EPC colony formation were found to be reduced in type 2 diabetic patients
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suffering from high circulating fatty acids(3). The studies from our group also showed
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that palmitate (PA), one of the most common FFAs, impaired adherence, migration
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and tube formation capacity of EPCs(13). However, molecular mechanisms of
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inhibitory effects of PA on EPCs have not yet been clearly addressed.
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Ceramide, one of the key FFAs metabolites, is an important bioactive molecule
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participating in a number of physiological and pathophysiological events (20). Studies
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have revealed that ceramide as a potentially important molecule mediating cellular
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dysfunction induced by PA in multiple cell types. For example, PA blocked insulin
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activation of PI3k/PKB by promoting the accumulation of ceramide and
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diacylglycerol in 3T3-L1 adipocytes and C2C12 myocytes (7). Here, we hypothesized
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that ceramide as a potential intermediate molecule, is responsible for PA-induced
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EPCs dysfunction through inhibiting AKT/eNOS signaling pathway. Our findings
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might shed lights on development of effective treatments for disorders with impaired
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EPCs.
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Materials and Methods
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EPCs Isolation and Culture
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The study was approved by Independent Ethics Committee of Tongji Hospital
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affiliated with Tongji University. Peripheral blood was collected from healthy adult
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volunteers and informed consent was signed prior to the study. EPCs isolation
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protocol was modified from previous studies (21, 23, 27) and established in our
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laboratory (13). In brief, peripheral blood mononuclear cells (PBMCs) from healthy
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volunteers were isolated by lymphoprep (1.077 g/mL) density barrier centrifugation.
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The low-density fraction (＜1.077 g/mL) was carefully removed from the interface
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and washed 3 times with PBS containing 2% FBS. Immediately after isolation, the
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cells were counted and 8×106 cells were plated on fibronectin-coated 12-well plates
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containing 1 mL Medium 199 (Invitrogen, Carlsbad, CA), supplemented with 20%
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fetal bovine serum, 100 U/mL penicillin/streptomycin (Invitrogen), 0.05 mg/mL
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bovine pituitary extract (Invitrogen), 10 μg/mL epidermal growth factor (Invitrogen),
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50 μg/mL gentamicine (Invitrogen), 50 μg/mL amphotericin-B (Invitrogen), 1 μg/mL
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hydrocortisone (Sigma), and 10 ng/mL human vascular endothelial growth factor 165
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(Sigma). Before use, the medium was passed through a 0.2-μm filter. After 4 days in
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culture, the nonadherent cells were removed and fresh medium was added. The
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medium was changed on day 6 and cells were kept in culture until day 7. The EPCs
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phenotype was confirmed by the presence of endothelial markers and the uptake of
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1,19-dioctadecyl-3,39-tetramethylindo-carbocyanine-labeled acetyl low-density
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lipoprotein (Di-Ac-LDL) and binding of Ulex europaeus agglutinin (UEA-1) as
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described previously. To detect the uptake of Di-Ac-LDL, cells were incubated with
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Di-Ac-LDL (2.4 mg/mL) at 37°C for 1 hour. Cells were then fixed with 2%
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paraformaldehyde for 10 minutes, and lectin staining was performed by incubation
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with fluorescein isothiocyanate (FITC)–labeled Ulex europaeus agglutinin I (lectin,
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10 mg/mL; Sigma) for 1 hour. After the staining, samples were viewed with an
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inverted fluorescent microscope. Dual-stained cells positive for both lectin and
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Di-Ac-LDL were judged to be EPCs. Two to three independent investigators
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evaluated the number of EPCs per well by counting 3 randomly selected high-power
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fields. To detect the expression of marker proteins, EPCs were detached with 1
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mmol/L EDTA in PBS, followed by repeated gentle flushing through a pipette tip.
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Cells were incubated for 15 minutes in the dark with monoclonal antibodies against
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human KDR, the FITC-labeled monoclonal antibody against human CD34, the
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phycoerythrin-conjugated monoclonal antibody against human CD133.
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Isotype-identical antibodies served as controls (IgG1-phycoerythrin and IgG2a-FITC,).
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Each analysis included 60,000 events.
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Preparation of media containing fatty acid-albumin complexes
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Saturated PA used in this study was purchased from Sigma. Lipid containing media
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was prepared by conjugating FFA to BSA. Briefly, PA was dissolved in ethanol at
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200 mmol/L, and then combined with 10% FFA-free low endotoxin BSA to a
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concentration range of 1-10 mmol/L. All stock solutions were adjusted to a pH value
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of 7.5, filter sterilized and stored at -20 °C. Control solution containing ethanol and
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BSA without PA was similarly prepared. Fresh working solutions were prepared by
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diluting stock solution (1:10) in Medium 199 supplemented with 2% FCS or 0.5%
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FCS as appropriate. All the final PA media contained 1% BSA while the ratio of FFA
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to BSA varied depending on the concentrations of PA.
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Ceramide assay
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Ceramide isolation protocol was established from our previous study (29).
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EPCs were incubated at 37 °C for 30 min in serum-free Medium 199. Then they were
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detached by 0.05% trypsin/0.53 mM EDTA (Gibco-BRL) and pelleted by
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centrifugation. The pellet was washed three times with 10 ml of cold PBS, and
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centrifuged again. The washed cells (5×106) were resuspended in 220 μL of cold 0.25
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M sucrose in PBS, transferred into a microtube, and disrupted by sonication. After the
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cell lysate was centrifuged at 800 × g for 10 min, the supernatant containing the
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cytosolic fraction with the cytoplasm and organelles was withdrawn. The pellet
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containing the membrane fraction was then washed three times with 1 ml of cold PBS,
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centrifuged, and suspended in 220 μL of PBS. Lipid extractions and ceramide
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determinations from each of above fractions were carried out according to the method
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of Zhou et al. (30). Briefly, 200 μL sample of each fraction was mixed with 4 mL
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chloroform/methanol (2:1) and extracted for its lipid contents for 30 min. After the
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addition of 1 mL water, the sample was vortexed and centrifuged. The lower phase
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containing the lipids was collected and evaporated to dryness under a nitrogen stream.
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The dry lipids were dissolved in 100 μL chloroform and reacted with 100 mM NAP,
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100 mM DCC and 100 mM DMAP (10 μl each) at −20°C for 3 h. After evaporation
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of the solvent under a nitrogen stream, the dry residue was suspended in 15 μL
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chloroform, then mixed with 2 mL hexane and centrifuged.The supernatant was
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removed, then vigorously mixed with 5 ml methanol/water (4:1) and centrifuged
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again. Fraction of 1 mL upper phase was collected and filtered through a 0.45-μm
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membrane filter. Twenty microliter filtrate was injected into an Econosphere CN 5μ
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column (4.6×250 mm, Alltech) in a HPLC (High performance liquid chromatography)
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system equipped with a Model 600 solvent delivery system (Waters), a Model 474
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fluorescence detector (Waters) and a Model 805 data station (Waters). The derivatized
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ceramide was separated from the by-products with 3% 2-propanol in n-hexane as the
182
mobile phase. The flow rate was 2.0 mL/min, and the eluted compounds were
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monitored by a fluorescence spectrophotometer at the excitation wavelength of 230
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nm and emission wavelength of 352 nm.
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Tube-formation Assay
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An ECM gel (Sigma) was thawed at 4 °C overnight and placed on a 96-well culture
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plate at 37 °C for 1 h to allow solidification. EPCs treated without or with PA were
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harvested and replated (10,000 cells/well) on the top of the solidified ECM gel in
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EBM-2 medium supplemented with 0.5% BSA and VEGF (100 ng/mL). Cells were
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incubated at 37 °C for 12 h. Tube formation was defined as a structure exhibiting a
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length four times its width. The networks of tubes were photographed from six
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randomly chosen fields with a microscope. The total length of the tube structures in
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each photograph was measured using Adobe Photoshop software (Adobe, San Jose,
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CA).
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Adhesion Assay
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After detachment and centrifugation, EPCs were resuspended in adhesion buffer
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(0.5% BSA in EBM-2), and identical numbers of cells were replated onto
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fibronectin-coated 24-well culture plates, incubated for 30 min at 37 °C, and then
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washed three times carefully with adhesion buffer to remove nonadherent cells.
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Adherent cells were counted by independent blinded investigators. Six independent
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fields were assessed for each well, and the average number of adherent cells was
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determined.
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Migration Assay
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Migration of EPCs was performed in a Transwell Chamber(Corning Costar, NewYork,
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NY). Briefly, EPCs were gently detached, harvested, and resuspended in chemotaxis
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buffer(EBM-2, 0.5% BSA). One hundred mL of chemotaxis buffer containing 1×105
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cells was added to the upper compartment, and 600 mL of chemotaxis buffer with 100
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ng/mL VEGF was added to the lower compartment. After incubation at 37 °C for 4 h,
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the filters were removed and cells in the lower compartment were counted using flow
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cytometry with appropriate gating for 20 sec at a high flow rate. Migratory rate was
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expressed as the percentage of input cells migrating into the lower chamber. All
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groups of experiments were performed in triplicate.
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Proliferaiton assay
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EPCs were isolated and incubated with different concentrations of palmitate for 48 h.
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Vybrant® MTT Cell Proliferation Assay (Life Technologies) was performed for EPC
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proliferation activity, The cell numbers were normalized to cells incubated in control
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medium.
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Western Blot Analysis
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EPCs were harvested in Western blot lysis buffer and the lysates were cleared by
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centrifugation at 12,000 × g for 10 min at 4 °C. The proteins were separated by
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10% SDS-PAGE and transferred to polyvinylidene difluoride membranes, then
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probed with one of the following primary antibodies against t-Akt, t-eNOS, p-Akt,
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p-eNOS, iNOS. All these antibodies were polyclonal antibodies from Santa Cruz
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Biotechnology (Santa Cruz, CA). The primary antibodies bound to the target proteins
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were then detected by horseradish peroxidase-conjugated anti-rabbit IgG (Promega,
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Madison, WI) and visualized with enhanced chemiluminescent detection (Pierce
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Biotechnology, Rockford, IL). Intensities of all target bands were normalized with
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that of the protein loading control GAPDH band calculated by the FluorChem 8900
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software system (Alpha Innotech, San Leandro, CA) (16).
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Determination of NO Generation
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NO is unstable, but it produces the stable end products nitrite and nitrate. Hence, the
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best index of NO generation is the sum of nitrite and nitrate. Culture media were
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harvested and stored at - 80 oC until used for the assays. Levels of nitrite and nitrate
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were measured as described previously(14). Briefly, nitrate was converted to nitrite
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with nitrate reductase, and total nitrite was reacted to the Griess reagent.
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Absorbance of the color product was determined at 540 nm with a spectrophotometer.
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Statistical analysis
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Datawere generally expressed as mean±SD. SPSS software version 11. 0 (SPSS, Inc.,
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Chicago, IL) was used for statistical analyses. Statistical significance among mean
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values was evaluated by one-way ANOVA tests for measurement data, and LSD-t test
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for comparison between each other. Differences were considered significant when p
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value was P＜0.05.
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Results
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PA induces EPCs dysfunction
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In order to determine the effects of PA on EPCs, we tested the adherence, migration
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and tube formation abilities of EPCs with or without PA treatments. Adhesion of
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EPCs to the extracellular matrix is important for EPCs mediated blood vessel
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formation. When treated with PA, EPCs exhibited impaired adhesion to fibronectin.
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PA inhibited adhesion at a concentration as low as 0.2 mM (Figure 1A). Migration is
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another critical function for EPCs, to assess the effect of PA on EPCs migration, we
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tested the migratory activity of EPCs in response to VEGF. The effect of PA on EPCs
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migration was evaluated using a modified Boyden chamber assay with VEGF as a
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chemoattract factor. As shown in Fig. 1A, VEGF-induced augmentation of EPCs
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migration was significantly inhibited in the PA treated group as compared with the
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control group. Finally, in vitro tube formation assay was also performed. As shown in
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Fig. 1B and 1C, the capacity for tube formation of EPCs on ECM gel was
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significantly reduced with PA treatment. The effect of PA on proliferation of EPCs
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was also determined by MTT assay, as shown in Fig. 1D, PA can inhibit proliferation
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of EPCs in a dose dependent manner. Taken together, our data suggested that PA
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treatment impaired normal functions for EPCs. Since there was no obvious difference
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in inhibitory effect of PA on EPCs functions between concentrations of 0.4 mM and
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0.6 mM. We used 0.4 mM PA for the following studies.
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PA inhibits Akt/eNOS pathway and decreases NO Production in EPCs
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Akt/eNOS pathway plays a pivotal role in the regulation of EPCs functions. We
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therefore investigated the effects of PA on Akt/eNOS signal pathway in EPCs.
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Immunoblotting showed that PA treatment inhibited Akt activation as evidenced by
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the reduction of specific phosphorylated (activated) Akt at the serine 473 and
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Threonine 308 phosphorylation sites in a time-dependent manner (Fig. 2A).
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It has been shown that FFAs can inhibit eNOS activity by reducing posttranslational
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phosphorylation at its Ser1177 site in endothelial cells (ECs) (29). To determine
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whether PA had any effect on eNOS activity in EPCs, we examined the eNOS
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activation by measuring its phosphorylation at Ser1177. As shown in Fig. 2B,
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significant reduction of eNOS phosphorylation was observed with PA treatment in a
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time-dependent manner. PA inhibited eNOS phosphorylation as soon as 15min at a
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concentration of 0.4 mM. Since Akt/eNOS signal pathway was inhibited by PA, we
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further assessed the effect of PA on NO production. In deed, we found reduction of
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NO was associated with reduction of phosphorylated eNOS (Fig 2B and 2D). Since
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both eNOS and inducible NOS (iNOS) can regulate NO production, we also checked
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the effects of PA on iNOS expression (Fig. 2C). We found PA treatment failed to alter
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iNOS expression, suggesting the selective inhibition of eNOS activity by PA (Figure
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2B and C).
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Ceramide mediates PA inhibitory effects on the Akt/eNOS pathway
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We have found that PA induces accumulation of ceramide, which mediates
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detrimental effects of PA in other cell types, such as endothelial cells (29). Thus, we
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hypothesized that PA would induce ceramide accumulation in EPCs. In deed, as
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shown in Fig. 3A, PA induced ceramide accumulation for 4-fold over basal levels in
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EPCs.
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Based on this finding, we further hypothesized that ceramide was a principal factor
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responsible to the inhibitory effects of PA on EPCs. To manipulate intracellular
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ceramide levels in EPCs, we relied on the use of a number of enzyme inhibitors to
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block ceramide synthesis. Ceramide biosynthesis requires the coordinate action of two
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enzymes, serine palmitoyltransferase and ceramide synthase. Serine
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palmitoyltransferase catalyzes the initial step, which involves the condensation of
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serine and palmitoyl-CoA to form 3-keto-sphinganine. Ketosphinganine is a
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sphingolipid that is subsequently reduced to form the sphingoid base sphinganine.
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Ceramide synthase catalyzes sphinganine acylation, producing dihydroceramide,
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which is then converted to ceramide by the introduction of a trans-4,5 double bond in
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the sphinganine moiety. Pretreating EPCs with myriocin, a fungal toxin that inhibits
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serine palmitoyltransferase, completely prevented the PA-induced increase in
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ceramide levels. Fumonisin B1, a fungal toxin that inhibits ceramide synthase, also
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abolished the PA effect on ceramide (Figure 3D). These results suggested that PA
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induced ceramide accumulation and this was mediated by the actions of the two
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enzymes responsible for the ceramide biosynthesis. To test the effects of ceramid on
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EPCs, cells were co-treated with PA and inhibitors, we found ceramide synthesis
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inhibitors abolished inhibition of Akt/eNOS signal pathway by PA (Figure 3 B, C),
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indicating ceramide might mediate PA-induced dysfunction of EPCs.
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Ceramide metabolite does not contribute to EPCs dysfunction
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It has been demonstrated that ceramide can be rapidly deacylated and glucosylated to
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give rise to a broad array of sphingolipid-derived molecules. Thus, one question
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remained. Is ceramide itself or, its metabolite, the principal mediator of the inhibitory
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effects of PA? To answer this question, we treated cells with inhibitor of ceramide
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deacylation, N-oleoylethanolamine (NOE), that have been shown previously to
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increase endogenous ceramide levels by blocking its metabolism. Treating EPCs with
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NOE increased cellular ceramide to the level comparable with PA treated alone
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(Figure 3D), and NOE markedly improved the inhibition of phosphorylated Akt by PA
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(Figure 3B, C) but did not affected the total protein expression of Akt and eNOS.
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Thus, we demonstrated that PA induces accumulation of ceramide, which appears to
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mediate PA inhibitory effects on the Akt/eNOS pathway. Furthermore, function of
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eNOS was tested by NO generation, consistent to the effects on eNOS
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phorsphorylation, we found block of ceramide synthesis could attenuation inhibitory
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effects of PA on generation of NO, while block of ceramide metabolism could further
339
inhibit NO production (Figure 3D).
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NO production rescues ceramide meditated EPCs dysfunction
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Since ceramide mediates the inhibition effect of PA on Akt /eNOS pathway and
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downstream NO production. Next, we asked whether ceramide inhibited EPCs
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through Akt/eNOS pathway, or it can directly work on EPCs. To test this hypothesis, a
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NO donor, SNP, was used. As shown in Fig. 4B and 4C, pretreatment with SNP
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significantly restored the ability of EPCs for adherence, migration(Figure 4A) and
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tube formation, suggesting that the inhibitory effect of ceramide on EPCs is through
347
Akt/eNOS pathway.
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Discussion
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In the present study, we showed that Akt/eNOS phosphorylation was reduced after PA
351
treatment, which hampered normal bioavailability of NO and led to impaired
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adherence, migration and tube formation capacity of EPCs. In addition, our results
353
demonstrated that PA induced accumulation of ceramide in EPCs. The detrimental
354
effects of PA on EPCs were mediated by ceramide, not its metabolites, via the
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inhibition of the Akt/eNOS pathway.
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The effect of PA on ceramide accumulation in different cell types was not extensively
357
studied. We showed previously that the PA caused ceramide accumulation in
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endothelial cells (29). It has demonstrated that grown in medium with endothelial cell
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growth supplement, EPCs express endothelial specific markers and show endothelial
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biological characters (15). Thus, we hypothesized that PA may induced ceramide
361
accumulation in EPCs. In this study we demonstrated that PA treatment significantly
362
increased ceramide concentration in EPCs. To minimize the possibility that our
363
observations were the result of nonspecific pharmacological effects, we employed
364
inhibitors capable of blocking separate enzymes in the various pathways. First, the
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fungal toxins myriocin and FB1 inhibit separate enzymes that are required for de novo
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ceramide synthesis (19, 25) and both protected EPCs from the accumulation of
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ceramide induced by PA. Second, NOE, an inhibitor of ceramidase (4), was able to
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aggravated the effect of PA ceramide accumulation. Thus, by using various inhibitors,
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our data indicated that PA can induce accumulation of ceramide in EPCs.
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Akt, a serine/threonine kinase, regulates several biological processes including
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cellular growth, proliferation and survival in multiple organs (18). Akt regulates EPCs
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functions and survival through phosphorylation/activation eNOS. Established
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evidence indicates Akt /eNOS pathway plays a pivotal role in the regulation of EPCs
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(10). In turn, eNOS modulates EPCs mobilization, migration, and survival via the
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generation of NO(10). In an ex vivo study, the migration and tube formation ability of
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early and late EPCs were related to NO (17). In line with these findings, our results
377
showed that the Akt/eNOS pathway plays an important role in the PA-induced EPCs
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dysfunction. Adherence, migration and tube formation capacities, eNOS and Akt
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phosphorylation as well as NO synthesized by EPCs were decreased by PA treatment.
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Furthermore, decreased NO production seems to play a pivotal role in adherence,
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migration and tube formation deficit, because pre-incubation of EPCs with SNP, an
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NO donor, restored proper of EPCs.
383
Nonetheless, the clear mechanism of PA regulating Akt/eNOS and NO release has not
384
been addressed. Studies indicated that ceramide accumulation with simultaneous
385
inhibition of Akt in cultured myotubes, L6 skeletal muscle cells, 3T3-L1 adipocytes
386
and cardiac myocytes exposed to PA (14, 24). On the basis of our results, PA induced
387
inactivation of Akt/eNOS pathway accompanying with accumulation of ceramide, we
388
hypothesized that ceramide might be one of the mechanisms that mediated the
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crosstalk PA and Akt/eNOS pathway.
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In humans, ceramides are collectively involved in physiological processes, such as
391
growth regulation and apoptosis, and in pathological conditions, such as diabetes and
392
cancers (11). Previously studies also showed that ceramides mediating a multitude of
393
distinct cellular signals (6). Our current study revealed the relationship of ceramide
394
accumulation and Akt/eNOS dephosphorylation in EPCs. However, we cannot study
395
every aspect of activities of PA-ceramide signaling in EPCs, for example, PA induced
396
accumulation of ceramide might also cause apoptosis in EPCs, therefore, suppress
397
EPCs functions; ceramide has also been shown to induce oxidative stresses in
398
endothelial cells, which might happen in EPCs as well. All of these possibilities will
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require further studies.
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In conclusion, the present study has revealed novel mechanism that PA induced
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accumulation of ceramide, which appears to mediate PA inhibitory effects on the
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Akt/eNOS pathway, leading to an overall dysfunction of EPCs. A better understanding
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of the regulation of EPCs function will enable the design of new pharmacological
404
therapies or regeneration strategies related to EPCs.
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Acknowledgments
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This work was supported by the National Natural Science Foundation of China (
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Grant No.81200244) to Xiaoyun Xie, by the Shanghai Natural Science Foundation
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(Grant No. 12ZR1428400) to Xiaoyun Xie, by Science and Technology Department
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of Hunan Province (Grant No.2013FJ3061) to Zhihong Li, Chenzhou Science,
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Technology Bureau (Grant No.CZ2013081) to Zhihong Li and by American Heart
416
Association (12SDG12070174) to Hua Zhu.
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Conflict of Interest
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The Authors declare no conflict of interests.
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Archives of medical research 39: 434-442, 2008. 14. Hage Hassan R, Bourron O, and Hajduch E. Defect of insulin signal in peripheral tissues: Important role of ceramide. World journal of diabetes 5: 244-257, 2014. 15. Hibbert B, Simard T, Ramirez FD, Pourdjabbar A, Raizman JE, Maze R, Wilson KR, Hawken S, and O'Brien ER. The effect of statins on circulating endothelial progenitor cells in humans: a systematic review. Journal of cardiovascular pharmacology 62: 491-496, 2013. 16. Jin F and Fondell JD. A novel androgen receptor-binding element modulates Cdc6 transcription in prostate cancer cells during cell-cycle progression. Nucleic acids research 37: 4826-4838, 2009. 17. Ma J, Both SK, Yang F, Cui FZ, Pan J, Meijer GJ, Jansen JA, and van den Beucken JJ. Concise review: cell-based strategies in bone tissue engineering and regenerative medicine. Stem cells translational medicine 3: 98-107, 2014. 18. Martelli AM, Tabellini G, Bressanin D, Ognibene A, Goto K, Cocco L, and Evangelisti C. The emerging multiple roles of nuclear Akt. Biochimica et biophysica acta 1823: 2168-2178, 2012. 19. Mehra VC, Jackson E, Zhang XM, Jiang XC, Dobrucki LW, Yu J, Bernatchez P, Sinusas AJ, Shulman GI, Sessa WC, Yarovinsky TO, and Bender JR. Ceramide-activated phosphatase mediates fatty acid-induced endothelial VEGF resistance and impaired angiogenesis. The American journal of pathology 184: 1562-1576, 2014. 20. Montefusco DJ, Chen L, Matmati N, Lu S, Newcomb B, Cooper GF, Hannun YA, and Lu X. Distinct signaling roles of ceramide species in yeast revealed through systematic perturbation and systems biology analyses. Science signaling 6: rs14, 2013. 21. Prokopi M, Pula G, Mayr U, Devue C, Gallagher J, Xiao Q, Boulanger CM, Westwood N, Urbich C, Willeit J, Steiner M, Breuss J, Xu Q, Kiechl S, and Mayr M. Proteomic analysis reveals presence of platelet microparticles in endothelial progenitor cell cultures. Blood 114: 723-732, 2009. 22. Raval Z and Losordo DW. Cell therapy of peripheral arterial disease: from experimental findings to clinical trials. Circulation research 112: 1288-1302, 2013. 23. Rustemeyer P, Wittkowski W, Jurk K, and Koller A. Optimized flow cytometric analysis of endothelial progenitor cells in peripheral blood. Journal of immunoassay & immunochemistry 27: 77-88, 2006. 24. Tomas E, Lin YS, Dagher Z, Saha A, Luo Z, Ido Y, and Ruderman NB. Hyperglycemia and insulin resistance: possible mechanisms. Annals of the New York Academy of Sciences 967: 43-51, 2002. 25. Tong M, Longato L, Ramirez T, Zabala V, Wands JR, and de la Monte SM. Therapeutic reversal of chronic alcohol-related steatohepatitis with the ceramide inhibitor myriocin. International journal of experimental pathology 95: 49-63, 2014. 26. Trombetta A, Togliatto G, Rosso A, Dentelli P, Olgasi C, Cotogni P, and Brizzi MF. Increase of palmitic acid concentration impairs endothelial progenitor cell and bone marrow-derived progenitor cell bioavailability: role of the
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STAT5/PPARgamma transcriptional complex. Diabetes 62: 1245-1257, 2013. 27. Vasa M, Fichtlscherer S, Adler K, Aicher A, Martin H, Zeiher AM, and Dimmeler S. Increase in circulating endothelial progenitor cells by statin therapy in patients with stable coronary artery disease. Circulation 103: 2885-2890, 2001. 28. Wojakowski W, Kucia M, Liu R, Zuba-Surma E, Jadczyk T, Bachowski R, Nabialek E, Kazmierski M, Ratajczak MZ, and Tendera M. Circulating very small embryonic-like stem cells in cardiovascular disease. Journal of cardiovascular translational research 4: 138-144, 2011. 29. Xiao-Yun X, Zhuo-Xiong C, Min-Xiang L, Xingxuan H, Schuchman EH, Feng L, Han-Song X, and An-Hua L. Ceramide mediates inhibition of the AKT/eNOS signaling pathway by palmitate in human vascular endothelial cells. Medical science monitor : international medical journal of experimental and clinical research 15: BR254-261, 2009. 30. Zhou Q, Zhang L, Fu XQ, and Chen GQ. Quantitation of yeast ceramides using high-performance liquid chromatography-evaporative light-scattering detection. Journal of chromatography B, Analytical technologies in the biomedical and life sciences 780: 161-169, 2002.
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Figure legends
544
Figure 1. Effect of PA on tube formation, adherence, migration capacities and
545
ceramide concentration of EPCs.
546
a dose dependent manner. Data were presented as mean ± SEM. (n=3 per each group,
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** p
1
Akt/eNOS Signaling Pathway Mediates Inhibition of Endothelial
2
Progenitor Cells by Palmitate-induced Ceramide
3
Minghuan Fu a,1, Zhihong Lib , Tao Tanc, Weixin Guod , Nanzi Xiee, Qing Liua, Hua
4
Zhu c* ,Xiaoyun Xiee*, Han Lei a*
5
a
6
Yuanjiagang District, Chongqing 400016, P.R. China
7
b
8
423000, China
9
c
The First Affiliated Hospital, Chongqing Medical University, No. 1 Youyi Road,
Division of General Surgery, Chenzhou First People′s Hospital, Chenzhou, Hunan
Department of Surgery, Davis Heart and Lung Research Institute, The Ohio State
10
University Wexner Medical Center, Columbus, Ohio 43210, the United States
11
d
12
of Medical sciences No. 106, Zhongshan Road, Guangzhou 510100, China
13
e
14
Shanghai 200065, China
15
Running Title: Ceramide mediates Palmitate-induced EPCs dysfunction
16
*Correspondence to:
17
Dr. Han Lei
18
The First Affiliated Hospital, Chongqing Medical University, No. 1 Youyi Road,
19
Yuanjiagang District, Chongqing 400016, P.R. China
20
Email: [email protected]
21
or Drs. Xiaoyun Xie, [email protected] and Hua Zhu, [email protected]
Guangdong Geriatrics Institute, Guangdong General Hospital, Guangdong Academy
Division of Geriatrics, Tongji Hospital, Tongji University, School of Medicine,
22
Copyright © 2014 by the American Physiological Society.
23
Abstract
24
Palmitate (PA) impairs endothelial progenitor cells (EPCs). However, the molecular
25
mechanism underlying the suppressive function of PA remains largely unknown.
26
Ceramide, a free fatty acid (FFAs) metabolite, mediates multiple cellular signals. We
27
hypothesized that ceramide acts as an intermediate molecule to mediate inhibition of
28
EPSc by PA. We first demonstrated that PA could inhibit attachment, migration and
29
tube formation of EPCs through suppression of Akt/eNOS signal pathway. In addition,
30
we observed PA could induce ceramide accumulation in EPCs. To test whether the
31
accumulation of ceramide causes EPCs dysfunction, ceramide synthesis inhibitors,
32
myriocin and fumonisin B1 (FB1) were used. We found both inhibitors could
33
effectively abolish PA-mediated EPCs inhibition. Furthermore, a ceramide
34
deacylation inhibitor, N-oleoylethanolamine (NOE), could augment the inhibitory
35
effect of PA on EPCs, indicating it is the ceramide, not its metabolites, that mediates
36
suppression of EPCs by PA. We previously showed that Akt/eNOS phosphorylation
37
was reduced after PA treatment, which in turn hampered normal bioavailability of
38
nitric oxide (NO) leading to impaired functions of EPCs. In order to test the role for
39
ceramide in this process, a clinically used NO donor, sodium nitroprusside (SNP), was
40
used. We found SNP could rescue the suppressive effects of ceramide on EPCs,
41
suggesting ceramide-mediated EPCs inhibition might be through reduction of NO
42
production. Taken together, our findings indicated that ceramide induced reduction of
43
NO might be the molecular mechanism for PA-mediated EPCs inhibition, thus,
44
targeting either ceramide or NO production might be an effective means for
45
improvement of EPCs functions in diseases.
46
Key words:
47
Palmitate, Ceramide, Endothelial Progenitor Cells, Stem Cells, Nitric Oxide
48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
67
Introduction
68
Endothelial progenitor cells (EPCs), were initially described and defined as a special
69
type of stem cell by Asahara et al(2). EPCs have been found in bone marrow, spleen,
70
umbilical cord blood (CB) and peripheral blood (PB), and can contribute to the
71
formation of new blood vessels in postnatal life and incorporate into injured vessels to
72
form mature endothelial cells (ECs) in response to tissue ischemia(9, 28). Increasing
73
evidences indicate that incorporation of EPCs play an important role in postnatal
74
neovascularization (1, 12).
75
In some pathological conditions such as diabetes and coronary artery disease, the
76
number of circulating EPCs is decreased and their functions are impaired (8, 22). As
77
previously highlighted, circulating EPCs in diabetes are subjected to many
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biochemical alterations attributable to the unfavorable vascular environment. Among
79
these factors, elevated levels of free fatty acids(FFAs) are commonly observed in
80
human patients with diabetes, obesity, and dyslipidemias. High FFA level is an
81
independent risk factor for atherosclerosis, hypertension, and sudden cardiac death(5).
82
FFAs may create a state of continuous and progressive damage to the circulating cells.
83
Therefore, FFAs may be one of the factors that influences growth and bioactivity of
84
EPCs. In deed, it has been reported that the ability of diabetic EPCs to integrate into
85
vascular networks was reduced after FFAs treatment(26). In addition, circulating
86
EPCs and EPC colony formation were found to be reduced in type 2 diabetic patients
87
suffering from high circulating fatty acids(3). The studies from our group also showed
88
that palmitate (PA), one of the most common FFAs, impaired adherence, migration
89
and tube formation capacity of EPCs(13). However, molecular mechanisms of
90
inhibitory effects of PA on EPCs have not yet been clearly addressed.
91
Ceramide, one of the key FFAs metabolites, is an important bioactive molecule
92
participating in a number of physiological and pathophysiological events (20). Studies
93
have revealed that ceramide as a potentially important molecule mediating cellular
94
dysfunction induced by PA in multiple cell types. For example, PA blocked insulin
95
activation of PI3k/PKB by promoting the accumulation of ceramide and
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diacylglycerol in 3T3-L1 adipocytes and C2C12 myocytes (7). Here, we hypothesized
97
that ceramide as a potential intermediate molecule, is responsible for PA-induced
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EPCs dysfunction through inhibiting AKT/eNOS signaling pathway. Our findings
99
might shed lights on development of effective treatments for disorders with impaired
100
101
102
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106
107
EPCs.
108
Materials and Methods
109
EPCs Isolation and Culture
110
The study was approved by Independent Ethics Committee of Tongji Hospital
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affiliated with Tongji University. Peripheral blood was collected from healthy adult
112
volunteers and informed consent was signed prior to the study. EPCs isolation
113
protocol was modified from previous studies (21, 23, 27) and established in our
114
laboratory (13). In brief, peripheral blood mononuclear cells (PBMCs) from healthy
115
volunteers were isolated by lymphoprep (1.077 g/mL) density barrier centrifugation.
116
The low-density fraction (＜1.077 g/mL) was carefully removed from the interface
117
and washed 3 times with PBS containing 2% FBS. Immediately after isolation, the
118
cells were counted and 8×106 cells were plated on fibronectin-coated 12-well plates
119
containing 1 mL Medium 199 (Invitrogen, Carlsbad, CA), supplemented with 20%
120
fetal bovine serum, 100 U/mL penicillin/streptomycin (Invitrogen), 0.05 mg/mL
121
bovine pituitary extract (Invitrogen), 10 μg/mL epidermal growth factor (Invitrogen),
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50 μg/mL gentamicine (Invitrogen), 50 μg/mL amphotericin-B (Invitrogen), 1 μg/mL
123
hydrocortisone (Sigma), and 10 ng/mL human vascular endothelial growth factor 165
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(Sigma). Before use, the medium was passed through a 0.2-μm filter. After 4 days in
125
culture, the nonadherent cells were removed and fresh medium was added. The
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medium was changed on day 6 and cells were kept in culture until day 7. The EPCs
127
phenotype was confirmed by the presence of endothelial markers and the uptake of
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1,19-dioctadecyl-3,39-tetramethylindo-carbocyanine-labeled acetyl low-density
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lipoprotein (Di-Ac-LDL) and binding of Ulex europaeus agglutinin (UEA-1) as
130
described previously. To detect the uptake of Di-Ac-LDL, cells were incubated with
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Di-Ac-LDL (2.4 mg/mL) at 37°C for 1 hour. Cells were then fixed with 2%
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paraformaldehyde for 10 minutes, and lectin staining was performed by incubation
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with fluorescein isothiocyanate (FITC)–labeled Ulex europaeus agglutinin I (lectin,
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10 mg/mL; Sigma) for 1 hour. After the staining, samples were viewed with an
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inverted fluorescent microscope. Dual-stained cells positive for both lectin and
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Di-Ac-LDL were judged to be EPCs. Two to three independent investigators
137
evaluated the number of EPCs per well by counting 3 randomly selected high-power
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fields. To detect the expression of marker proteins, EPCs were detached with 1
139
mmol/L EDTA in PBS, followed by repeated gentle flushing through a pipette tip.
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Cells were incubated for 15 minutes in the dark with monoclonal antibodies against
141
human KDR, the FITC-labeled monoclonal antibody against human CD34, the
142
phycoerythrin-conjugated monoclonal antibody against human CD133.
143
Isotype-identical antibodies served as controls (IgG1-phycoerythrin and IgG2a-FITC,).
144
Each analysis included 60,000 events.
145
Preparation of media containing fatty acid-albumin complexes
146
Saturated PA used in this study was purchased from Sigma. Lipid containing media
147
was prepared by conjugating FFA to BSA. Briefly, PA was dissolved in ethanol at
148
200 mmol/L, and then combined with 10% FFA-free low endotoxin BSA to a
149
concentration range of 1-10 mmol/L. All stock solutions were adjusted to a pH value
150
of 7.5, filter sterilized and stored at -20 °C. Control solution containing ethanol and
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BSA without PA was similarly prepared. Fresh working solutions were prepared by
152
diluting stock solution (1:10) in Medium 199 supplemented with 2% FCS or 0.5%
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FCS as appropriate. All the final PA media contained 1% BSA while the ratio of FFA
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to BSA varied depending on the concentrations of PA.
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Ceramide assay
156
Ceramide isolation protocol was established from our previous study (29).
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EPCs were incubated at 37 °C for 30 min in serum-free Medium 199. Then they were
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detached by 0.05% trypsin/0.53 mM EDTA (Gibco-BRL) and pelleted by
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centrifugation. The pellet was washed three times with 10 ml of cold PBS, and
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centrifuged again. The washed cells (5×106) were resuspended in 220 μL of cold 0.25
161
M sucrose in PBS, transferred into a microtube, and disrupted by sonication. After the
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cell lysate was centrifuged at 800 × g for 10 min, the supernatant containing the
163
cytosolic fraction with the cytoplasm and organelles was withdrawn. The pellet
164
containing the membrane fraction was then washed three times with 1 ml of cold PBS,
165
centrifuged, and suspended in 220 μL of PBS. Lipid extractions and ceramide
166
determinations from each of above fractions were carried out according to the method
167
of Zhou et al. (30). Briefly, 200 μL sample of each fraction was mixed with 4 mL
168
chloroform/methanol (2:1) and extracted for its lipid contents for 30 min. After the
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addition of 1 mL water, the sample was vortexed and centrifuged. The lower phase
170
containing the lipids was collected and evaporated to dryness under a nitrogen stream.
171
The dry lipids were dissolved in 100 μL chloroform and reacted with 100 mM NAP,
172
100 mM DCC and 100 mM DMAP (10 μl each) at −20°C for 3 h. After evaporation
173
of the solvent under a nitrogen stream, the dry residue was suspended in 15 μL
174
chloroform, then mixed with 2 mL hexane and centrifuged.The supernatant was
175
removed, then vigorously mixed with 5 ml methanol/water (4:1) and centrifuged
176
again. Fraction of 1 mL upper phase was collected and filtered through a 0.45-μm
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membrane filter. Twenty microliter filtrate was injected into an Econosphere CN 5μ
178
column (4.6×250 mm, Alltech) in a HPLC (High performance liquid chromatography)
179
system equipped with a Model 600 solvent delivery system (Waters), a Model 474
180
fluorescence detector (Waters) and a Model 805 data station (Waters). The derivatized
181
ceramide was separated from the by-products with 3% 2-propanol in n-hexane as the
182
mobile phase. The flow rate was 2.0 mL/min, and the eluted compounds were
183
monitored by a fluorescence spectrophotometer at the excitation wavelength of 230
184
nm and emission wavelength of 352 nm.
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Tube-formation Assay
186
An ECM gel (Sigma) was thawed at 4 °C overnight and placed on a 96-well culture
187
plate at 37 °C for 1 h to allow solidification. EPCs treated without or with PA were
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harvested and replated (10,000 cells/well) on the top of the solidified ECM gel in
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EBM-2 medium supplemented with 0.5% BSA and VEGF (100 ng/mL). Cells were
190
incubated at 37 °C for 12 h. Tube formation was defined as a structure exhibiting a
191
length four times its width. The networks of tubes were photographed from six
192
randomly chosen fields with a microscope. The total length of the tube structures in
193
each photograph was measured using Adobe Photoshop software (Adobe, San Jose,
194
CA).
195
Adhesion Assay
196
After detachment and centrifugation, EPCs were resuspended in adhesion buffer
197
(0.5% BSA in EBM-2), and identical numbers of cells were replated onto
198
fibronectin-coated 24-well culture plates, incubated for 30 min at 37 °C, and then
199
washed three times carefully with adhesion buffer to remove nonadherent cells.
200
Adherent cells were counted by independent blinded investigators. Six independent
201
fields were assessed for each well, and the average number of adherent cells was
202
determined.
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Migration Assay
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Migration of EPCs was performed in a Transwell Chamber(Corning Costar, NewYork,
205
NY). Briefly, EPCs were gently detached, harvested, and resuspended in chemotaxis
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buffer(EBM-2, 0.5% BSA). One hundred mL of chemotaxis buffer containing 1×105
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cells was added to the upper compartment, and 600 mL of chemotaxis buffer with 100
208
ng/mL VEGF was added to the lower compartment. After incubation at 37 °C for 4 h,
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the filters were removed and cells in the lower compartment were counted using flow
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cytometry with appropriate gating for 20 sec at a high flow rate. Migratory rate was
211
expressed as the percentage of input cells migrating into the lower chamber. All
212
groups of experiments were performed in triplicate.
213
Proliferaiton assay
214
EPCs were isolated and incubated with different concentrations of palmitate for 48 h.
215
Vybrant® MTT Cell Proliferation Assay (Life Technologies) was performed for EPC
216
proliferation activity, The cell numbers were normalized to cells incubated in control
217
medium.
218
Western Blot Analysis
219
EPCs were harvested in Western blot lysis buffer and the lysates were cleared by
220
centrifugation at 12,000 × g for 10 min at 4 °C. The proteins were separated by
221
10% SDS-PAGE and transferred to polyvinylidene difluoride membranes, then
222
probed with one of the following primary antibodies against t-Akt, t-eNOS, p-Akt,
223
p-eNOS, iNOS. All these antibodies were polyclonal antibodies from Santa Cruz
224
Biotechnology (Santa Cruz, CA). The primary antibodies bound to the target proteins
225
were then detected by horseradish peroxidase-conjugated anti-rabbit IgG (Promega,
226
Madison, WI) and visualized with enhanced chemiluminescent detection (Pierce
227
Biotechnology, Rockford, IL). Intensities of all target bands were normalized with
228
that of the protein loading control GAPDH band calculated by the FluorChem 8900
229
software system (Alpha Innotech, San Leandro, CA) (16).
230
Determination of NO Generation
231
NO is unstable, but it produces the stable end products nitrite and nitrate. Hence, the
232
best index of NO generation is the sum of nitrite and nitrate. Culture media were
233
harvested and stored at - 80 oC until used for the assays. Levels of nitrite and nitrate
234
were measured as described previously(14). Briefly, nitrate was converted to nitrite
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with nitrate reductase, and total nitrite was reacted to the Griess reagent.
236
Absorbance of the color product was determined at 540 nm with a spectrophotometer.
237
238
Statistical analysis
239
Datawere generally expressed as mean±SD. SPSS software version 11. 0 (SPSS, Inc.,
240
Chicago, IL) was used for statistical analyses. Statistical significance among mean
241
values was evaluated by one-way ANOVA tests for measurement data, and LSD-t test
242
for comparison between each other. Differences were considered significant when p
243
value was P＜0.05.
244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259
260
Results
261
PA induces EPCs dysfunction
262
In order to determine the effects of PA on EPCs, we tested the adherence, migration
263
and tube formation abilities of EPCs with or without PA treatments. Adhesion of
264
EPCs to the extracellular matrix is important for EPCs mediated blood vessel
265
formation. When treated with PA, EPCs exhibited impaired adhesion to fibronectin.
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PA inhibited adhesion at a concentration as low as 0.2 mM (Figure 1A). Migration is
267
another critical function for EPCs, to assess the effect of PA on EPCs migration, we
268
tested the migratory activity of EPCs in response to VEGF. The effect of PA on EPCs
269
migration was evaluated using a modified Boyden chamber assay with VEGF as a
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chemoattract factor. As shown in Fig. 1A, VEGF-induced augmentation of EPCs
271
migration was significantly inhibited in the PA treated group as compared with the
272
control group. Finally, in vitro tube formation assay was also performed. As shown in
273
Fig. 1B and 1C, the capacity for tube formation of EPCs on ECM gel was
274
significantly reduced with PA treatment. The effect of PA on proliferation of EPCs
275
was also determined by MTT assay, as shown in Fig. 1D, PA can inhibit proliferation
276
of EPCs in a dose dependent manner. Taken together, our data suggested that PA
277
treatment impaired normal functions for EPCs. Since there was no obvious difference
278
in inhibitory effect of PA on EPCs functions between concentrations of 0.4 mM and
279
0.6 mM. We used 0.4 mM PA for the following studies.
280
PA inhibits Akt/eNOS pathway and decreases NO Production in EPCs
281
Akt/eNOS pathway plays a pivotal role in the regulation of EPCs functions. We
282
therefore investigated the effects of PA on Akt/eNOS signal pathway in EPCs.
283
Immunoblotting showed that PA treatment inhibited Akt activation as evidenced by
284
the reduction of specific phosphorylated (activated) Akt at the serine 473 and
285
Threonine 308 phosphorylation sites in a time-dependent manner (Fig. 2A).
286
It has been shown that FFAs can inhibit eNOS activity by reducing posttranslational
287
phosphorylation at its Ser1177 site in endothelial cells (ECs) (29). To determine
288
whether PA had any effect on eNOS activity in EPCs, we examined the eNOS
289
activation by measuring its phosphorylation at Ser1177. As shown in Fig. 2B,
290
significant reduction of eNOS phosphorylation was observed with PA treatment in a
291
time-dependent manner. PA inhibited eNOS phosphorylation as soon as 15min at a
292
concentration of 0.4 mM. Since Akt/eNOS signal pathway was inhibited by PA, we
293
further assessed the effect of PA on NO production. In deed, we found reduction of
294
NO was associated with reduction of phosphorylated eNOS (Fig 2B and 2D). Since
295
both eNOS and inducible NOS (iNOS) can regulate NO production, we also checked
296
the effects of PA on iNOS expression (Fig. 2C). We found PA treatment failed to alter
297
iNOS expression, suggesting the selective inhibition of eNOS activity by PA (Figure
298
2B and C).
299
Ceramide mediates PA inhibitory effects on the Akt/eNOS pathway
300
We have found that PA induces accumulation of ceramide, which mediates
301
detrimental effects of PA in other cell types, such as endothelial cells (29). Thus, we
302
hypothesized that PA would induce ceramide accumulation in EPCs. In deed, as
303
shown in Fig. 3A, PA induced ceramide accumulation for 4-fold over basal levels in
304
EPCs.
305
Based on this finding, we further hypothesized that ceramide was a principal factor
306
responsible to the inhibitory effects of PA on EPCs. To manipulate intracellular
307
ceramide levels in EPCs, we relied on the use of a number of enzyme inhibitors to
308
block ceramide synthesis. Ceramide biosynthesis requires the coordinate action of two
309
enzymes, serine palmitoyltransferase and ceramide synthase. Serine
310
palmitoyltransferase catalyzes the initial step, which involves the condensation of
311
serine and palmitoyl-CoA to form 3-keto-sphinganine. Ketosphinganine is a
312
sphingolipid that is subsequently reduced to form the sphingoid base sphinganine.
313
Ceramide synthase catalyzes sphinganine acylation, producing dihydroceramide,
314
which is then converted to ceramide by the introduction of a trans-4,5 double bond in
315
the sphinganine moiety. Pretreating EPCs with myriocin, a fungal toxin that inhibits
316
serine palmitoyltransferase, completely prevented the PA-induced increase in
317
ceramide levels. Fumonisin B1, a fungal toxin that inhibits ceramide synthase, also
318
abolished the PA effect on ceramide (Figure 3D). These results suggested that PA
319
induced ceramide accumulation and this was mediated by the actions of the two
320
enzymes responsible for the ceramide biosynthesis. To test the effects of ceramid on
321
EPCs, cells were co-treated with PA and inhibitors, we found ceramide synthesis
322
inhibitors abolished inhibition of Akt/eNOS signal pathway by PA (Figure 3 B, C),
323
indicating ceramide might mediate PA-induced dysfunction of EPCs.
324
Ceramide metabolite does not contribute to EPCs dysfunction
325
It has been demonstrated that ceramide can be rapidly deacylated and glucosylated to
326
give rise to a broad array of sphingolipid-derived molecules. Thus, one question
327
remained. Is ceramide itself or, its metabolite, the principal mediator of the inhibitory
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effects of PA? To answer this question, we treated cells with inhibitor of ceramide
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deacylation, N-oleoylethanolamine (NOE), that have been shown previously to
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increase endogenous ceramide levels by blocking its metabolism. Treating EPCs with
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NOE increased cellular ceramide to the level comparable with PA treated alone
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(Figure 3D), and NOE markedly improved the inhibition of phosphorylated Akt by PA
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(Figure 3B, C) but did not affected the total protein expression of Akt and eNOS.
334
Thus, we demonstrated that PA induces accumulation of ceramide, which appears to
335
mediate PA inhibitory effects on the Akt/eNOS pathway. Furthermore, function of
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eNOS was tested by NO generation, consistent to the effects on eNOS
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phorsphorylation, we found block of ceramide synthesis could attenuation inhibitory
338
effects of PA on generation of NO, while block of ceramide metabolism could further
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inhibit NO production (Figure 3D).
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NO production rescues ceramide meditated EPCs dysfunction
341
Since ceramide mediates the inhibition effect of PA on Akt /eNOS pathway and
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downstream NO production. Next, we asked whether ceramide inhibited EPCs
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through Akt/eNOS pathway, or it can directly work on EPCs. To test this hypothesis, a
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NO donor, SNP, was used. As shown in Fig. 4B and 4C, pretreatment with SNP
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significantly restored the ability of EPCs for adherence, migration(Figure 4A) and
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tube formation, suggesting that the inhibitory effect of ceramide on EPCs is through
347
Akt/eNOS pathway.
348
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Discussion
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In the present study, we showed that Akt/eNOS phosphorylation was reduced after PA
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treatment, which hampered normal bioavailability of NO and led to impaired
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adherence, migration and tube formation capacity of EPCs. In addition, our results
353
demonstrated that PA induced accumulation of ceramide in EPCs. The detrimental
354
effects of PA on EPCs were mediated by ceramide, not its metabolites, via the
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inhibition of the Akt/eNOS pathway.
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The effect of PA on ceramide accumulation in different cell types was not extensively
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studied. We showed previously that the PA caused ceramide accumulation in
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endothelial cells (29). It has demonstrated that grown in medium with endothelial cell
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growth supplement, EPCs express endothelial specific markers and show endothelial
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biological characters (15). Thus, we hypothesized that PA may induced ceramide
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accumulation in EPCs. In this study we demonstrated that PA treatment significantly
362
increased ceramide concentration in EPCs. To minimize the possibility that our
363
observations were the result of nonspecific pharmacological effects, we employed
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inhibitors capable of blocking separate enzymes in the various pathways. First, the
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fungal toxins myriocin and FB1 inhibit separate enzymes that are required for de novo
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ceramide synthesis (19, 25) and both protected EPCs from the accumulation of
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ceramide induced by PA. Second, NOE, an inhibitor of ceramidase (4), was able to
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aggravated the effect of PA ceramide accumulation. Thus, by using various inhibitors,
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our data indicated that PA can induce accumulation of ceramide in EPCs.
370
Akt, a serine/threonine kinase, regulates several biological processes including
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cellular growth, proliferation and survival in multiple organs (18). Akt regulates EPCs
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functions and survival through phosphorylation/activation eNOS. Established
373
evidence indicates Akt /eNOS pathway plays a pivotal role in the regulation of EPCs
374
(10). In turn, eNOS modulates EPCs mobilization, migration, and survival via the
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generation of NO(10). In an ex vivo study, the migration and tube formation ability of
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early and late EPCs were related to NO (17). In line with these findings, our results
377
showed that the Akt/eNOS pathway plays an important role in the PA-induced EPCs
378
dysfunction. Adherence, migration and tube formation capacities, eNOS and Akt
379
phosphorylation as well as NO synthesized by EPCs were decreased by PA treatment.
380
Furthermore, decreased NO production seems to play a pivotal role in adherence,
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migration and tube formation deficit, because pre-incubation of EPCs with SNP, an
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NO donor, restored proper of EPCs.
383
Nonetheless, the clear mechanism of PA regulating Akt/eNOS and NO release has not
384
been addressed. Studies indicated that ceramide accumulation with simultaneous
385
inhibition of Akt in cultured myotubes, L6 skeletal muscle cells, 3T3-L1 adipocytes
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and cardiac myocytes exposed to PA (14, 24). On the basis of our results, PA induced
387
inactivation of Akt/eNOS pathway accompanying with accumulation of ceramide, we
388
hypothesized that ceramide might be one of the mechanisms that mediated the
389
crosstalk PA and Akt/eNOS pathway.
390
In humans, ceramides are collectively involved in physiological processes, such as
391
growth regulation and apoptosis, and in pathological conditions, such as diabetes and
392
cancers (11). Previously studies also showed that ceramides mediating a multitude of
393
distinct cellular signals (6). Our current study revealed the relationship of ceramide
394
accumulation and Akt/eNOS dephosphorylation in EPCs. However, we cannot study
395
every aspect of activities of PA-ceramide signaling in EPCs, for example, PA induced
396
accumulation of ceramide might also cause apoptosis in EPCs, therefore, suppress
397
EPCs functions; ceramide has also been shown to induce oxidative stresses in
398
endothelial cells, which might happen in EPCs as well. All of these possibilities will
399
require further studies.
400
In conclusion, the present study has revealed novel mechanism that PA induced
401
accumulation of ceramide, which appears to mediate PA inhibitory effects on the
402
Akt/eNOS pathway, leading to an overall dysfunction of EPCs. A better understanding
403
of the regulation of EPCs function will enable the design of new pharmacological
404
therapies or regeneration strategies related to EPCs.
405 406 407 408 409
410
Acknowledgments
411
This work was supported by the National Natural Science Foundation of China (
412
Grant No.81200244) to Xiaoyun Xie, by the Shanghai Natural Science Foundation
413
(Grant No. 12ZR1428400) to Xiaoyun Xie, by Science and Technology Department
414
of Hunan Province (Grant No.2013FJ3061) to Zhihong Li, Chenzhou Science,
415
Technology Bureau (Grant No.CZ2013081) to Zhihong Li and by American Heart
416
Association (12SDG12070174) to Hua Zhu.
417
Conflict of Interest
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The Authors declare no conflict of interests.
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Figure legends
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Figure 1. Effect of PA on tube formation, adherence, migration capacities and
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ceramide concentration of EPCs.
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a dose dependent manner. Data were presented as mean ± SEM. (n=3 per each group,
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** p
