Published online before print December 20, 2007, doi: 10.1161/ATVBAHA.107.157339
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© 2008 American Heart Association, Inc.
Cell Biology/Signaling |
Oleate Induces a Myofibroblast-Like Phenotype in Mesangial Cells
From the Division of Nephrology and Hypertension, Department of Medicine, Case Western Reserve University and University Hospital Case Medical Center, Cleveland Ohio.
Correspondence to Michael S. Simonson, Department of Medicine, Division of Nephrology, Biomedical Research Building, Rm. 427, Case Western Reserve University, 2109 Adelbert Road, Cleveland, OH 44106. E-mail mss5{at}po.cwru.edu
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Abstract |
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Objective— High circulating free fatty acids, commonly associated with obesity and insulin resistance, impair structure and function of the microvasculature. However, the mechanisms by which fatty acids cause microvascular remodeling are unclear. Using the mesangial cell model of microvascular pericytes, we demonstrate that the monounsaturated free fatty acid oleate induces a myofibroblast phenotype, an important cell fate transition in fibrotic remodeling of the extracellular matrix.
Materials and Results— Oleate induced a time- and dose-dependent increase in secretion of collagen I and fibronectin. Oleate also induced the myofibroblast phenotype markers 
smooth muscle actin and ED-A fibronectin, and the magnitude of marker protein expression was similar to that for transforming growth factor (TGF)-β. Oleate raised TGF-β secretion 2.2-fold, and processing of latent to bioactive TGF-β was also elevated. Oleate rapidly stimulated extracellular signal-regulated kinase1/2, and a pharmacological MEK inhibitor blocked TGF-β secretion and conversion to the myofibroblast phenotype. A neutralizing TGF-β antibody and a TGF-β receptor kinase inhibitor blocked oleate-induced collagen I, smooth muscle actin, and ED-A fibronectin, suggesting that oleate-stimulated TGF-β was necessary for inducing myofibroblasts.
Conclusions— Collectively, these results demonstrate that oleate can induce a myofibroblast phenotype in mesangial cells, which suggests a mechanism whereby elevated free fatty acids might promote microvascular remodeling in vivo.
Here, we demonstrate that the free fatty acid oleate induces a myofibroblast phenotype in microvascular pericytes. This oleate-induced conversion suggests a mechanism whereby elevated free fatty acids could affect a profibrotic shift in pericyte phenotype and microvascular remodeling.
Key Words: mesangial cells • microvascular • pericytes • free fatty acids • TGF-β; fibrosis
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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The microvasculature, comprised of arterioles, capillaries, and venules, is surrounded by pericytes that provide structural support and control vascular permeability, contractility, and angiogenesis.1–4 Pericytes are pleuripotent, and phenotypic transition of microvascular pericytes is observed in microangiopathy of diabetes,5,6 hypertension,1,3,7 scleroderma,8,9 and perhaps in calcification of blood vessels.10,11 Adaptive phenotypic transitions in pericytes are thought to be important in microvascular dysfunction and remodeling in response to injury.2,4
A recently described fate transition in pericytes is conversion to myofibroblasts,8,9,12,13 which produces profibrotic effector cells that remodel the extracellular matrix.14–16 Myofibroblasts secrete excess extracellular matrix proteins such as fibrillar collagens I and III and the fibronectin EDA-FN splice variant. By assembling stress fibers rich in smooth muscle actin (SMA) that link to the extracellular matrix through fibronexus adhesion complexes, myofibroblasts contract and remodel the extracellular matrix into a fibrotic lesion.14,16 In the context of fibrosis, the myofibroblast phenotype is driven by paracrine and autocrine signaling mechanisms by cytokines and growth factors, such as transforming growth factor (TGF)-β17 and endothelin-1,18 and by biophysical forces such as interstitial fluid flow.19
In obesity and conditions of insulin resistance, the microvasculature is commonly exposed to high concentrations of circulating free fatty acids (FFAs).20,21 Elevated circulating FFA, and perhaps intracellular deposition of fat in the microvasculature, impairs microvascular function and structure by mechanisms that are not well-understood.22–24 High FFA is associated with microvascular remodeling in the liver and kidney in disorders such as nonalcoholic fatty liver disease and diabetic nephropathy.25,26 Myofibroblast phenotypic conversions have been reported in the liver and kidney of rodents fed high-fat diets and in hypertriglyceridemic rat kidneys,25–28 but the extent to which pericytes developed the myofibroblast phenotype was not assessed in these studies. It is unknown whether exposure of pericytes to high FFA induces the myofibroblast phenotype.
The objective of this study was to determine whether FFAs induce the myofibroblast conversion of microvascular pericytes. Here, we demonstrate that the monounsaturated FFA oleate induces the myofibroblast phenotype in cultured mesangial cells, a model for pericyte conversion to myofibroblasts.3,29,30 We focused on oleate because it is one of the most abundant FFAs in human plasma.20 Moreover oleate resists autoxidation,31 which minimizes the confounding effect of FFA oxidation on vascular cell function. Our current findings suggest that oleate can induce myofibroblasts by a TGF-β–dependent mechanism.
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Materials and Methods |
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Reagents
Recombinant human TGF-β1 was from R&D Systems. SB 431542 was from Sigma, PD98059 was from Cell Signaling, and U0126 was from EMD Biosciences. Antibodies were as follows: affinity-purified rabbit anti-human collagen type I (Biodesign International); collagen IV and fibronectin (Rockland);
SMA (Epitomics); myosin IIA (Sigma); mouse monoclonal ED-A FN (Sigma); mouse monoclonal neutralizing TGF-β antisera (R&D). Mouse clone E10 anti–phospho-ERK1/2 (PThr202/PTyr204), mouse anti-total ERK2 clone 3A7, PD98059, and U0126 were from Cell Signaling Technology.
Preparation of FFA-Albumin Complexes
Fatty acid-albumin complexes were prepared by the protocol of Spector32 as previously described.33 Briefly, a sodium salt of oleate, linoleate and palmitate (
-Chek Prep) were dissolved in PBS and warmed to increase solubility without damaging the fatty acid.32 The clear oleate salt solution was complexed to 5% fatty acid-free BSA in PBS at a 6:1 fatty acid to BSA molar ratio. The sterile filtered fatty acid-albumin solution was added to 0.5% serum-containing cell culture medium to obtain the final concentration. Fatty acid concentration in the medium was confirmed with an enzymatic colorimetric assay (NEFA C, Wako). The fatty acid-BSA complex did not significantly alter the culture media pH. The normal physiological ratio of FFA to albumin is approximately 2:1, but serum FFA levels are higher in patients with obesity, yielding ratios of 6:1 or higher.34,35 We therefore used oleate to albumin ratios of 6:1 to mimic the FFA-albumin complexes found in obesity. The FFA-albumin complexes contained less than 0.15 ng/mL endotoxin (LAL Endotoxin Test, Cell Sciences Inc), which did not elevate TGF-β secretion in mesangial cells. Immunoreactive TGF-β (see below) was undetectable in the fatty acid-albumin complexes.
Human Mesangial Cell Culture and Viability Assay
Human mesangial cells (HMC) were obtained from Cambrex Bioscience Inc (Walkersville, Md) and were cultured and maintained as described previously.36,37 Briefly, cells were positive for desmin, vimentin, and myosin IIA, but did not stain for factor VIII, keratin, or common leukocyte antigen. For cell viability assays, cells in 24-well plates were exposed to oleate for 24 hours and 48 hours, and MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) reduction was measured for 30 minutes at 37°C using a protocol from the manufacturer (Promega Inc, G3580). Absorbance at 490 nm was recorded in 96-well plates using a SpectraMax 190 microplate reader (Molecular Devices). Wells with media alone served as the blank. For neutral lipid staining, cells on coverslips were fixed with 2% paraformaldehyde and stained with oil red O as previously described.38
Induction of Myofibroblast Markers
Cells in 60-mm plates at 80% confluence were made quiescent for 24 hours in DMEM with 0.5% FBS, then stimulated with fatty acid or TGF-β for 24 or 48 hours in the presence of 50 µg/mL β-aminopropionitrile to minimize cross-linking. The supernatant was collected and the monolayer was solubilized in a 5 mol/L guanidine-0.1 mol/L Tris buffer (pH 8.6) with protease inhibitors as described.39 All samples were stored immediately at –80°C. ELISA for secreted collagen I, collagen IV, and fibronectin (FN) in the medium was as described.40,41 ELISA was also used to measure cell-associated ED-A FN and SMA.42 One hundred µL of media or cell extracts were absorbed to Nunc Maxisorp 96-well plates overnight at 4°C. Nonspecific binding was blocked in 1.0% BSA, 100 mmol/L phosphate buffer, pH 8.2. The wells were then incubated with a primary antibody, the appropriate affinity purified and biotin conjugated goat anti-IgG and HRP-conjugated streptavidin. A solution of 3,5,2',5'-tetramethylbenzidine was added followed by quenching with acid and an absorbance reading at 450 nm. All values were in the linear range and were normalized for cell number.
Immunofluorescent Analysis of SMA and ED-A FN
Quiescent cells on glass coverslips were fixed in methanol:acetone at –20°C. After blocking with 3.0% BSA in TBS-T for 1 hour, primary antibody (1:500 for ED-A FN or 1:300 for SMA) was added overnight at 4°C. The primary antibody was detected using highly cross-absorbed goat anti-mouse or rabbit IgG labeled with ALEXA Fluor 488, and the monolayer was mounted in MAXfluor with DAPI (Lake Placid Biologicals). Images were acquired with a SPOT RT camera (Diagnostic Instruments) at the same exposure to facilitate semiquantitative comparison.
Measurements of Total TGF-β
Quiescent cells were treated with oleate-albumin complexes or lipopolysaccharide (LPS; Sigma) as a positive control for TGF-β secretion. The cell culture supernatant was collected, and total TGF-β in acidified media (to release TGF-β from latent complexes) was determined by ELISA for TGF-β1 (R&D).
Measurements of Latent TGF-β Activation
Conditioned media from mesangial cells exposed to oleate or BSA alone was added to mink lung cells stably transfected with a minimal promoter from the plasminogen activator inhibitor (PAI)-1 gene linked to luciferase as previously described.43 After 24 hours in conditioned media the cytosolic luciferase activity was measured as described.44 The results were expressed relative to TGF-β activity with media alone. Recombinant human TGF-β1 was used as the standard,43 and the specificity was assessed by adding a neutralizing TGF-β antisera (10 µg/mL) as indicated.
Western Blotting for PhosphoERK1/2 in Cell Lysates
Cell monolayers solubilized in CHAPS extraction buffer (50 mmol/L Pipes/HCI, pH 6.5, 2 mmol/L EDTA, 0.1% Chaps, 20 µg/mL leupeptin, 10 µg/mL pepstatin A, 10 µg/mL aprotinin, 5 mmol/L DTT, 2 mmol/L Na pyrophosphate, 1 mmol/L Na3VO4, and 1 mmol/L NaF) were analyzed by Western blotting as previously described45,46 using an antibody that recognizes dually phosphorylated ERK1/2 (PThr202/PTyr204). Membranes were reprobed for total ERK2 to confirm equal protein loading.
Measurement of Steady State Col11, Col12 mRNA
Quantitiative real-time RT-PCR was used to measure mRNA transcripts in total RNA (RNeasy, Qiagen) isolated from mesangial cells.38,41 Five µg of total RNA was used to synthesize cDNA with a T7-(dT)24 primer (Genset) and RT Superscript II (GIBCO BRL) for 1 hour at 42°C. Primers for human mRNA sequences were designed using Primer3 (v. 4.0) available at http://fokker.wi.mit. edu/primer3/input.htm. Primer sequences were: human Col11 (Genbank Accession number NM_000088) upstream: GTG CTA AAG GTG CCA ATG GT; downstream: ACC AGG TTC ACC GCT GTT AC; human Col12 (Genbank Accession number NM_000089) upstream: AGG AGT TGT TGG ACC ACA GG; downstream: CGT CCT CTC TCA CCA GGA AG; GAPDH: upstream: TGT CCC CAC TGC CAA CGT GT; downstream: AGG GTA CTT TAT TGA TGG TA. Real Time-PCR was performed using a Stratagene MX3000P machine for 40 cycles as follows: 30 s at 95°C, 30 s at 68°C, and 30 s at 72°C preceded by 10 minutes of incubation at 95°C. Using agarose gel electrophoresis of the PCR products, we confirmed that only 1 band of the predicted molecular weight was present. A melting curve recorded at the end of the reaction was used for correction of the amplification curve. The amount of collagen I mRNA was determined and expressed relative to GAPDH mRNA in the same sample by the 2-
CT method.47
Statistics
Data are mean±SD from at least n=3 independent experiments. Statistical significance was calculated by the unpaired Student t test for single comparisons or by ANOVA followed by a Bonferroni post-hoc test for multiple comparisons as appropriate using InStat (GraphPad). *P<0.05.
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Results and Discussion |
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Oleate Induces a Myofibroblast Phenotype in Cultured Mesangial Cells
Mesangial cells are microvascular pericytes (ie, mural cells) from the renal glomerulus, and cultured cells from glomerular explants are a well-established in vitro model of pericyte biology.4,29,30 Mesangial cells in vivo are exposed to circulating FFA because the glomerular endothelium is highly fenestrated, and intracellular fatty acid deposition in mesangial cells has been observed in humans and in animal models of obesity and diabetes (see26 for review). Furthermore, elevation of circulating oleic acid-albumin complexes increases the number of
SMA-positive myofibroblasts in the glomerulus and tubulointerstitium of a rat model of protein-overload nephropathy,48 suggesting that increments in circulating oleate can induce myofibroblasts in vivo. To determine whether exposure of cells to oleate was cytotoxic, MTS reduction was measured in cells treated for 24 and 48 hours. There was no increase in MTS reduction in cells exposed to media alone or to albumin (Figure 1A). However, 24 hours after adding oleate, MTS reduction was significantly elevated; at 48 hours MTS reduction nearly doubled, suggesting that cells exposed to 200 µmol/L oleate were viable. Similar results were observed with 400 µmol/L oleate (data not shown). Oil red O staining revealed that exposure to oleate greatly increased the frequency and size of neutral lipid droplets in the cytosol (Figure 1B and inset).
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A crucial aspect of myofibroblast conversion is secretion of collagen I and assembly of a fibrotic, fibrillar extracellular matrix. We therefore asked whether oleate elevates collagen I secretion in quiescent cells. Oleate stimulated collagen I secretion 1.5-fold at 24 hours and 1.9-fold at 48 hours (Figure 2A). Albumin alone did not raise collagen I secretion above basal levels. Significantly, the polyunsaturated FFA linoleate and the saturated FFA palmitate raised collagen I secretion 48 hour after addition (Figure 2A, 48-hour data only), suggesting that FFA other than oleate can induce collagen I. Collagen I secretion in oleate-treated cells was dose-responsive (Figure 2B), and the effective doses of oleate were well below the plasma level of FFA in obesity and insulin resistance.20 As measured by quantitative RT-PCR, oleate increased mRNA expression for Col12 (6.1-fold versus albumin at 24 hours) more than Col11 (Figure 2C). In diabetes the phenotypic transition of mesangial cells involves increased secretion of collagen IV and fibronectin,49 and we found that secretion of both extracellular matrix proteins was significantly elevated by oleate (Figure 2D).
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The prototypical markers of the myofibroblast phenotype are smooth muscle actin (SMA) and the ED-A FN splice variant,14,15 which are both associated with the development of fibrotic lesions in vivo. To definitively determine whether oleate induces myofibroblast conversion, we assessed induction of SMA and ED-A FN in cultured mesangial cells. After 48-hour exposure to oleate, the FFA induced SMA expression 2.6-fold compared with 2.5-fold induction by the established stimulus for the myofibroblast phenotype, TGF-β, at a maximal dose (Figure 3A). Oleate also induced the myofibroblast marker ED-A FN, although induction of ED-A FN by oleate was less than that for TGF-β (Figure 3A). Immunofluorescent staining showed that oleate-induced SMA was localized to cytoskeletal bundles typical of stress fibers in myofibroblasts (Figure 3B and 3C). Oleate also raised immunoreactive ED-A FN (Figure 3D and 3E), and the distribution of staining was similar to that observed in fibroblasts stimulated to become myofibroblasts with TGF-β.14,15 Significant immunofluorescence was not observed in cells stained with the secondary antibody alone (Figure 3F and 3G). Collectively, the experiments in this section suggest that oleate is a potent activator of myofibroblast-like changes in mesangial cells.
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Oleate Stimulates TGF-β Secretion and Processing
We next investigated the mechanism by which oleate induces myofibroblast conversion of cultured mesangial cells. TGF-β is a well-established stimulus for myofibroblasts in vitro and in vivo, and induction of myofibroblasts by TGF-β plays a critical role in fibrosis.14 In addition, rat renal TGF-β immunoreactivity increases when the level of circulating oleate-albumin complexes is elevated.48 We thus asked whether oleate increases TGF-β secretion. Oleate was added to quiescent mesangial cells, and total TGF-β in the supernatant was measured by ELISA. Oleate increased TGF-β secretion at 24 hours, and the concentration of TGF-β remained elevated at 48 hours (Figure 4A); albumin alone did not increase TGF-β. The increase in TGF-β secretion by oleate was similar to LPS, a potent stimulus for TGF-β.
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Cells secrete TGF-β as latent complexes in which the 25-kDa mature, active TGF-β associates noncovalently with its propeptide and a latent TGF-β binding protein.50 TGF-β latent complexes cannot bind to TGF-β receptors, and so the complex sequesters TGF-β–induced signaling. On latent TGF-β activation, bioactive TGF-β dissociates from these complexes, providing a rapid mechanism to increase local concentrations of TGF-β. We asked whether oleate increases latent TGF-β activation. Bioactive TGF-β in cell supernates was measured with a reporter cell line that responds only to the processed, bioactive form of TGF-β.43,50 After 24 hours with oleate, the amount of active TGF-β in the conditioned medium was 182.3 pg/mL. Active TGF-β was 2.3-fold higher in conditioned media from oleate-treated cells (Figure 4A). Preadsorption of TGF-β by a neutralizing antisera demonstrated that the assay was specific for TGF-β in the conditioned media. These results suggest that oleate stimulates latent TGF-β activation in mesangial cells.
TGF-β Secretion and Myofibroblast Induction in Oleate-Treated Cells Requires ERK1/2
We next investigated the signal transduction mechanism by which oleate stimulates TGF-β secretion and the myofibroblast phenotype in mesangial cells. Oleate has previously been shown to stimulate ERK1/2 in ECV-304 endothelial cells51 and in aortic smooth muscle cells.52 To determine whether oleate also activates ERK1/2 in mesangial cells, quiescent cells were treated with oleate and ERK1/2 activity was detected by Western blotting with a phosphospecific antibody that recognizes phosphorylation of the activating epitope (ie, PThr202/PTyr204). Oleate rapidly stimulated phosphorylation of ERK1/2 (Figure 5A). Phosphorylation was elevated at 15 minutes and remained high at 60 minutes. Immunoblotting of total ERK2 demonstrated equivalent protein loading (Figure 5A). Oleate-stimulated ERK1/2 phosphorylation was markedly attenuated by pretreatment with the MEK inhibitor U0126 (Figure 5A).
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To determine whether oleate-activated ERK1/2 is required for raising TGF-β, we pretreated quiescent cells with U0126 or PD98059 and used ELISA to measure TGF-β secretion in the supernatant. As expected, oleate elevated TGF-β at 24 and 48 hours, and oleate-induced TGF-β secretion was blocked by U0126 and PD98059 (Figure 5B), demonstrating a requirement for ERK1/2. U0126 and PD98059 also prevented latent TGF-β activation in response to oleate (Figure 5B). Additional experiments showed that activation of ERK1/2 was required for induction of myofibroblast phenotype markers by oleate. Oleate elevated collagen I secretion 2.1-fold, which was blocked by U0126 (Figure 5C). The MEK inhibitors similarly inhibited SMA and ED-A FN induction by oleate in mesangial cells (Figure 5C). The MEK inhibitors alone did not affect the basal level of collagen I, SMA, and ED-A FN in mesangial cells (data not shown). These results demonstrate that latent TGF-β activation and myofibroblast induction requires ERK1/2 in mesangial pericytes exposed to oleate.
Oleate Induces Myofibroblasts by a TGF-β–Based Mechanism
To determine whether oleate induction of myofibroblasts requires TGF-β, we studied the effects of a TGF-β–neutralizing antibody and a TGF-β receptor kinase inhibitor on induction of myofibroblast marker proteins. Preceding addition of oleate, quiescent cells were preincubated for 1 hour with a monoclonal antibody that recognizes and neutralizes the bioactivity of human TGF-β1, β2, and β3. The monoclonal antibody prevented induction of collagen I, SMA, and ED-A FN by oleate, suggesting that myofibroblast conversion requires TGF-β (Figure 6A). An irrelevant mouse IgG at the same concentration did not block induction of collagen I, SMA, or ED-A FN, suggesting that the inhibitory effect was specific for the neutralizing antibody. SB 431542 is a selective inhibitor of TGF-β type I activin receptor-like kinases.53 When quiescent cells were pretreated with SB 431542, oleate-induced collagen I, SMA, and ED-A FN were inhibited by 72%, 74%, and 67%, respectively (Figure 6B). The vehicle for SB43152 (0.01% DMSO) did not inhibit induction of myofibroblast markers by oleate. These results suggest that the oleate-induced myofibroblast phenotype in mesangial cells requires TGF-β activity.
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In summary, our results suggest that FFA induce myofibroblasts and matrix protein secretion in cultured microvascular pericytes. Moreover, oleate stimulates ERK1/2 activity in pericytes, which is required for the phenotypic conversion to myofibroblasts. In addition, oleate raises TGF-β by an ERK1/2-dependent mechanism that involves latent TGF-β activation. The increment of TGF-β is required for the oleate-induced myofibroblast phenotype. Further studies are necessary to explain how FFAs induce TGF-β and the myofibroblast phenotype in pericytes. One possibility is that intracellular uptake of FFA induces myofibroblasts, through metabolism of long-chain coenzyme A (CoA) species, formation of intracellular lipid storage droplets, or by serving as ligands for nuclear hormone receptors. Another possibility is that FFAs bind to and activate cell surface receptors that initiate signaling cascades, as has recently been reported for the G protein–coupled receptor platelet glycoprotein (GP)-4054 or the toll-like receptor 4.55 None of these pathways have been linked previously to induction of myofibroblasts.
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Acknowledgments |
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Sources of Funding
This work was supported by grants from the Rosenberg Foundation of the Centers for Dialysis Care (Cleveland, Ohio).
Disclosures
None.
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Footnotes |
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Original received June 1, 2007; final version accepted December 3, 2007.
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