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(Circulation Research. 1996;79:611-619.)
© 1996 American Heart Association, Inc.

Articles

Oleic Acid–Induced Mitogenic Signaling in Vascular Smooth Muscle Cells

A Role for Protein Kinase C

Gang Lu, Thomas A. Morinelli, Kathryn E. Meier, Steven A. Rosenzweig, Brent M. Egan

the Department of Cellular and Molecular Pharmacology, Medical University of South Carolina, Charleston.

Correspondence to Brent M. Egan, MD, Division of Clinical Pharmacology, Medical University of South Carolina, 171 Ashley Ave, CSB 826H, Charleston, SC 29425.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
As an initial step in testing the hypothesis that high oleic acid concentrations contribute to vascular remodeling in obese hypertensive patients by activating protein kinase C (PKC), the effects of oleic acid on primary cultures of rat aortic smooth muscle cells (RASMCs) were studied. Oleic acid, an 18-carbon cis-monounsaturated fatty acid (18:1 [cis]), from 25 to 200 µmol/L significantly increased [³H]thymidine uptake in RASMCs with an EC₅₀ of 41.0 µmol/L and a maximal response of 196±15% of control (P<.01). Oleic acid from 25 to 200 µmol/L caused a concentration-dependent increase in the number of RASMCs in culture at 6 days, reaching a maximum of 210±13% of control at 100 µmol/L (P<.001). PKC inhibition with 4 µmol/L bisindolylmaleimide I and PKC depletion (, µ, , and ) with 24-hour exposure to 200 nmol/L phorbol 12-myristate 13-acetate in RASMCs eliminated the mitogenic effects of oleic acid but did not reduce responses to 10% FBS. Stimulation of intact cells with oleic acid induced a peak increase of cytosolic PKC activity, reaching 328±8% of control (P<.001), but did not enhance PKC activity in the membrane fraction (105±4%, P=NS). The oleic acid–induced increase of PKC activity in cell lysates was similar in the presence and absence of Ca²⁺, phosphatidylserine, and diolein (maximum response, 360±4% versus 342±9% of control, P=NS). Unlike phorbol 12-myristate 13-acetate, oleic acid over 24 hours did not downregulate any of the four PKC isoforms detected in RASMCs. Oleic acid treatment activated mitogen-activated protein (MAP) kinase. PKC depletion in RASMCs eliminated the rise in thymidine uptake, activation of PKC, and activation of MAP kinase in response to oleic acid. In contrast to oleic acid, 50 to 200 µmol/L stearic (18:0) and elaidic (18:1 [trans]) acids, which are less effective activators of PKC than oleic acid, did not enhance thymidine uptake. These data suggest that oleic acid induces proliferation of RASMCs by activating PKC, particularly one or more of the Ca²⁺-independent isoforms, and raise the possibility that the higher oleic acid concentrations observed in obese hypertensive patients may contribute to vascular remodeling.

Key Words: oleic acid • protein kinase C • mitogenesis • vascular smooth muscle cell • vascular remodeling

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abdominal obesity is linked with a cluster of risk factors for atherosclerosis, including hypertension, diabetes, and dyslipidemia, with insulin resistance as a common denominator.^{1 2 3 4} The available evidence supports a link between resistance to insulin's nonesterified fatty acid–lowering action, activation of PKC, and vascular disease in those with the insulin resistance syndrome.^{5 6} We reported that obese hypertensive patients have higher nonesterified fatty acids than lean normotensive patients.⁷ OA, an 18-carbon lipid with one double bond in the cis position (18:1 [cis]), was the fatty acid with the highest concentration in plasma, and OA values were significantly greater in obese hypertensive patients (172±11 µmol/L) than in lean normotensive subjects (113±20 µmol/L). We then found that plasma fatty acid concentrations and turnover were greater during euglycemic hyperinsulinemic clamp studies in abdominally obese hypertensive patients than in either abdominally obese or lean normotensive subjects.⁸ Since elevations of plasma fatty acids during clamp studies^{9 10} are related to higher fatty acids over a 24-hour period and also after a mixed meal,^{11 12} obese hypertensive patients probably have higher plasma fatty acids at several points during the day compared with obese and lean normotensive individuals.

Nonesterified fatty acids, especially cis-unsaturates such as OA, directly activate PKC.^{13 14} In human platelets, OA is a more potent activator of the Ca²⁺-independent PKC isoforms, predominantly (EC₅₀, 5 µmol/L), than the Ca²⁺-dependent PKC isoforms, and ß (EC₅₀, 50 µmol/L).¹⁴ The EC₅₀ for activation of Ca²⁺-independent PKC by cis-unsaturated fatty acids is within the range of estimated intracellular fatty acid concentrations.¹⁵ Moreover, in platelets, OA fully activated the Ca²⁺-independent isoforms of PKC but caused only half-maximal stimulation of the Ca²⁺-dependent isoforms.¹⁴ These data raise the possibility that fatty acids are physiological regulators of Ca²⁺-independent PKC activity.

Activation of PKC can induce vascular smooth muscle cell proliferation.¹⁶ Thus, one might hypothesize that OA has a mitogenic effect in vascular smooth muscle cells by activating PKC. If this hypothesis were confirmed, it would raise the possibility that abnormalities of nonesterified fatty acids contribute to vascular remodeling and atherosclerosis in subjects with the insulin-resistance syndrome, which includes obese hypertensive patients. Consequently, we examined the mitogenic effects of selected 18-carbon fatty acids, with an emphasis on OA, in primary cultures of RASMCs. Measurements of [³H]thymidine incorporation, cell number, Ca²⁺-dependent and -independent PKC activity, and MAP kinase activity in response to OA were made under control conditions and after both PKC inhibition and depletion in primary cultures of RASMCs.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
NEFAs, including oleic, linoleic, elaidic, and stearic, were purchased from Sigma Chemical Co. Sodium salts were prepared by dissolving these NEFAs in 95% ethanol, followed by the addition of 0.5N NaOH. This mixture was evaporated under nitrogen, and the sodium salts of the fatty acids were reconstituted in water. Cell culture materials and PKC substrate peptide Ser²⁵PKC(19-31) were obtained from GIBCO BRL. Fatty acid–free albumin was obtained from ICN Biomedicals Inc. [³H]Thymidine (6.7 Ci/mmol, 37.0 MBq) and [-P³²]ATP (3000 Ci/mmol, 37.0 MBq) were provided by Du Pont NEN. ECL Western blotting kits were obtained from Amersham Corp. Monoclonal antibodies against specific isoforms of PKC and anti–phosphotyrosine-agarose conjugate were secured from Transduction Laboratories. DEAE-Sephacel and Bio-Spin chromatography columns were purchased from Sigma and Bio-Rad Laboratories, respectively.

Cell Culture
RASMCs were cultured by procedures modified from Chamley-Campbell et al.¹⁷ By use of a protocol approved by the Animal Research Committee, Sprague-Dawley rats (150 to 200 g) were killed instantly by decapitation. A 1-cm section of aorta was removed and placed in 1x DMEM. Adherent fat and connective tissue were gently removed with fine sterile forceps. The aorta was minced into small cube-shaped specimens and incubated with 1x DMEM/1 mg/mL collagenase for 1 hour. The individual pieces of vessel segments were seeded in a T-25 culture flask for at least 15 minutes to ensure adherence to the bottom surface. They were then incubated with 3 mL of 1x DMEM supplemented with 20% (vol/vol) FBS, 100 U/mL penicillin, and 100 U/mL streptomycin at 37°C in 95% air/5% CO₂. Approximately 7 to 10 days later, the segments were removed, and cells were placed into a 150-cm² flask, and the medium was changed to DMEM containing 10% FBS. Cells were characterized morphologically as smooth muscle by phase-contrast microscopy and by immunostaining with -actin.

[³H]Thymidine Uptake
Effects of OA on [³H]Thymidine Uptake in RASMCs
RASMCs were grown to subconfluence in 10% FBS in 24-well plates and then incubated in DMEM supplemented with 0.1% FBS for 48 hours to induce quiescence. OA (12.5 to 200 µmol/L) or 10% FBS was added to triplicate wells in fresh DMEM supplemented with 0.1% FBS and 100 µmol/L fatty acid–free albumin for 24 hours. The cells were pulse-labeled with [³H]thymidine (0.5 µCi per well) 6 hours before the completion of the 24-hour incubation period. The medium was removed, and the cell monolayer was washed sequentially with cold PBS (twice), 0.3 mL of cold 0.3 mol/L perchloric acid for 30 seconds, and cold PBS (twice). The cells were then solubilized by adding 0.1% SDS/0.1N NaOH (0.5 mL). The solubilized cell monolayer (0.4 mL) was added to 5 mL of Hydrofluor (National Diagnostics), and incorporation of [³H]thymidine was determined by liquid scintillation spectrometry. Incorporation was expressed as a percentage of that seen under the control basal condition (DMEM with 0.1% FBS and 100 µmol/L albumin).

Effect of PKC Inhibition and Depletion on [³H]Thymidine Uptake
Before the addition of OA and PMA, quiescent RASMCs were subjected to a 1-hour preincubation with various PKC inhibitors (eg, bisindolylmaleimide I) or a 24-hour preincubation with 200 nmol/L PMA to deplete PKC activity. The uptake of [³H]thymidine in response to OA (12.5 to 200 µmol/L), 10 nmol/L PMA, and 10% FBS was studied using the same experimental protocol described above.

Effect of Four Different 18-Carbon Fatty Acids on [³H]Thymidine Uptake
Oleic (18:1 [cis]), linoleic (18:2 [cis]), stearic (18:0), and elaidic (18:1 [trans]) acids were added to quiescent RASMCs for 24 hours. The effects of these four nonesterified fatty acids at identical concentrations ranging from 50 to 200 µmol/L on [³H]thymidine uptake were quantified as described above.

Effects of OA on RASMC Number
In a separate set of experiments, RASMCs were seeded at 12 500 cells per well, grown to subconfluence, growth-arrested by serum deprivation, and treated with different concentrations of OA, with or without 4 µmol/L bisindolylmaleimide I for 6 days. The media were changed after 72 hours and included the same concentration of OA and bisindolylmaleimide I every 72 hours. Cells were resuspended with 0.3 mL trypsin/EDTA (0.05%/0.5 mmol/L), and cell number was determined using a hemocytometer.

Western Blot Analysis
RASMCs were grown to confluence in 100-mm Petri dishes and incubated in 0.1% FBS/DMEM for 48 hours to induce quiescence. The monolayer was exposed to 200 nmol/L PMA in 0.1%/FBS DMEM for 10 minutes and 3, 6, 12, or 24 hours in 5% CO₂, at 37°C. The incubation was stopped by aspiration of medium, followed by three washes with ice-cold PBS. The cells were scraped into cold lysis buffer containing (mmol/L) Tris-HCl 20 (pH 7.5), ß-glycerophosphate 80, EDTA 5, EGTA 10, phenylmethylsulfonyl fluoride 1, dithiothreitol 2, and benzamidine 10. The suspended cells were homogenized by sonicating for 5 seconds and centrifuged at 100 000g for 20 minutes at 4°C. The supernatant was designated as the cytosolic fraction. The remaining pellet was resuspended in 0.4% Triton lysis buffer on ice for a minimum of 30 minutes and then sonicated for 10 seconds at 4°C. After a 1-minute centrifugation at 2000g, the supernatant was collected and designated as the membrane fraction. The protein contents were estimated by the Bradford protein assay.^{18 19}

Proteins in the cytosol, membrane extracts, and whole-cell lysate (10 µg per lane) were resolved by SDS-PAGE on 7.5% Laemmli gels, electrophoretically transferred to polyvinylidene membranes (Millipore), and then immunoblotted with monoclonal antibodies against the , ß, , , , , , µ, , and isoforms of PKC. Anti-mouse IgG horseradish peroxidase–conjugated antibody was used as the secondary antibody. Visualization of the blot was carried out using the ECL Western blotting system.

PKC Enzyme Activity
Confluent RASMCs in 100-mm Petri dishes were incubated with 0.1% FBS DMEM for 48 hours to induce quiescence. The monolayer was exposed to different concentrations of OA, PMA, and various PKC inhibitors in 0.1% FBS DMEM for 5 minutes. Cells were scraped and prepared as described above. The cytosolic and particulate fractions were used as the enzyme preparations. The assay for PKC activity (³²P transfer into substrate peptide) was modified from the procedure of Neary and colleagues^{20 21} and Yong.²² PKC substrate peptide Ser²⁵PKC(19-31) is a peptide (Arg-Phe-Ala-Arg-Lys-Gly-Ser-Leu-Arg-Gln-Lys-Asn-Val) derived from the pseudosubstrate region (in which an alanine is replaced by a serine). The peptide is among the best substrates for all the PKC isoenzymes.^{23 24 25} The reaction mixture contained 20 mmol/L HEPES, 150 µmol/L calcium chloride, 4 µmol/L PKC substrate peptide, 60 µg/mL phosphatidylserine, 6 µg/mL diolein, 50 µmol/L ATP (mixed with [-³²P]ATP, 10⁶ cpm), 10 mmol/L MgCl₂ (pH 7.5), and an appropriate amount of cytosolic and particulate protein in a final volume of 50 µL. Phosphorylation of PKC substrate peptide was initiated by adding the enzyme preparation and was terminated after 5 minutes by transferring 40 µL of the reaction mixture to a 2x2-cm square of Whatman phosphocellulose paper. The filter paper was placed immediately in 150 mmol/L phosphoric acid and rinsed three more times for 30 minutes each. After the final wash, papers were transferred to a scintillation vial for liquid scintillation counting. The PKC-dependent reaction was calculated as the difference between activity in the presence and absence (with 10 mmol/L EGTA) of Ca²⁺, phosphatidylserine, and diolein. Each enzyme preparation was analyzed in triplicate, and the results were expressed as picomoles ATP transferred per minute per milligram protein.

In addition, in vitro studies on direct activation of PKC by OA were performed. Whole-cell lysates from untreated RASMCs were partially purified using DEAE-Sephacel chromatography.²⁶ PKC activity was assessed in the presence and absence of Ca²⁺ using the protocol described above.

MAP Kinase Activity Assay
Confluent RASMCs in 100-mm Petri dishes were incubated in 0.1% FBS/DMEM for 48 hours. The monolayers with/without PKC depletion were then exposed to 100 µmol/L OA or 100 nmol/L PMA in serum-free DMEM for the appropriate period of time under 5% CO₂ at 37°C. The incubation was stopped by the addition of ice-cold PBS. The cells were scraped into PBS and then pelleted by centrifugation for 20 seconds (2000g). The pellet was suspended in cold lysis buffer (mmol/L: HEPES 20, ß-glycerophosphate 80, EDTA 2, EGTA 10, and dithiothreitol 2). Subsequently, the suspended pellet was sonicated for 5 seconds and centrifuged at 100 000g at 4°C. The supernatant was used as the enzyme preparation for assay of activated MAP kinase. The assay was performed by following established procedures with ³²P phosphorylation of myelin basic protein as a measurement of MAP kinase activity in cell lysate.²⁷ MAP kinase activity detected in the cytosolic extracts was corrected for protein and expressed as a percentage of the activity measured in untreated cells. In the same experimental condition, RASMCs with or without PKC depletion were exposed to 100 µmol/L OA or 100 nmol/L PMA for 10 minutes. The cell lysates were immunoprecipitated with anti–phosphotyrosine-agarose conjugate, resolved by SDS-PAGE, transferred to polyvinylidene membranes (Millipore), and then immunoblotted with anti–MAP kinase antibody. Densitometry was performed on these blots.

Statistical Analysis
Data are presented as mean±SEM. Data were analyzed with SPSS 6.0 (SPSS Inc). One-way ANOVA followed by Duncan's multiple range test was used to compare the cell number and PKC activity changes between treatment and control groups. The concentration-response curves were analyzed using sigmoidal dose-response relations, as described below, with Graphpad Prism 1.03 (Graphpad Software Inc) to define EC₅₀, where Y is bottom+(top-bottom)/(1+10^{log EC₅₀-X}), X is the logarithm of concentration, and Y is response. Values of P.05 were considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effects of OA on [³H]Thymidine Uptake in RASMCs
As shown in Fig 1, the addition of OA in concentrations ranging from 25 to 200 µmol/L to primary cultures of RASMCs induced a concentration-dependent increase of [³H]thymidine incorporation into acid-insoluble material, with an EC₅₀ of 41 µmol/L (95% confidence interval, 28 to 63 µmol/L). At a concentration of 200 µmol/L, OA increased thymidine uptake to values roughly double that of control, whereas 10% FBS increased thymidine uptake to values approximately three times that of control.

View larger version (21K): [in this window] [in a new window]   Figure 1. Effects of OA on [3H]thymidine uptake in RASMCs. Various concentrations of OA were added to quiescent RASMCs for 24 hours at 37°C. [3H]Thymidine uptake was measured 6 hours after addition of [3H]thymidine. The OA EC50 is depicted by the vertical line intercepting the abscissa. Data are presented as mean±SEM for three independent experiments in triplicate.

Effects of OA on RASMC Number
Under basal conditions (ie, 48 hours of growth arrest with 0.1% FBS, followed by a 6-day incubation with 0.1% FBS/DMEM and 100 µmol/L fatty acid–free albumin), there were 2.61±0.24x10⁴ cells per well (n=6). As shown in Table 1, the addition of 25 to 200 µmol/L OA for 6 days caused significant concentration-dependent increases in the number of cells compared with the control condition, whereas the 12.5 µmol/L concentration of OA did not significantly raise cell number. The increase in cell number in response to OA was completely abolished by pretreatment with 4 µmol/L bisindolylmaleimide I, a specific PKC inhibitor.

View this table: [in this window] [in a new window]   Table 1. Effects of OA on RASMC Number at 6 Days

Effects of PKC Inhibition and Depletion on OA-Induced [³H]Thymidine Uptake
PKC Inhibitors
Previous studies on human platelets and Swiss 3T3 fibroblasts have shown that bisindolylmaleimide I at concentrations of 5 µmol/L provides highly selective inhibition of PKC.^{28 29 30} In the present study, 100 µmol/L OA increased thymidine uptake to 188±8% of control. Preincubation of cells with 1, 2, and 4 µmol/L bisindolylmaleimide I before addition of 100 µmol/L OA lowered thymidine uptake progressively to 182±3%, 147±3%, and 101±5% of control, respectively. In contrast to the effect on OA-stimulated thymidine uptake, 4 µmol/L bisindolylmaleimide did not significantly reduce thymidine uptake in RASMCs stimulated with 10% FBS (298±10% versus 278±12%, P=NS). Other PKC inhibitors including 10 mmol/L polymyxin B, 10 nmol/L staurosporine, and 50 µmol/L H7 also abolished the increase of thymidine uptake in RASMCs to 82±6%, 75±9%, 69±11% of control, respectively. However, polymyxin B, staurosporine, and H7 also reduced basal thymidine uptake to 66±7%, 55±8%, 51±9% of control, respectively, whereas 4 µmol/L bisindolylmaleimide I did not (95±7% of control). These data on basal thymidine suggest that polymyxin B, staurosporine, and H7 have toxic effects on RASMCs. This impression was consistent with morphological evidence that concentrations of these compounds commonly used to inhibit PKC adversely affected cell morphology and viability, whereas 4 µmol/L bisindolylmaleimide I did not (data not shown).

PKC Depletion
RASMCs were treated with 200 nmol/L PMA for different lengths of time up to 24 hours. After 24 hours of exposure to 200 nmol/L PMA, no immunologically reactive PKC isoforms, including the two atypical isoforms, were detected in whole-cell lysates (Fig 2, top). PKC was the first to be downregulated and was absent within 6 hours of exposure of cells to PMA. PKCµ was absent by 12 hours, whereas the atypical isoforms required 24 hours for complete removal. In contrast to PMA, treatment with 100 µmol/L OA for 24 hours did not downregulate any of the four PKC isoforms detected in RASMCs (Fig 2, bottom). Depletion of PKC with PMA abolished the concentration-dependent increase of thymidine uptake in RASMCs stimulated with OA but not the response to 10% FBS, as shown in Fig 3.

View larger version (51K): [in this window] [in a new window]   Figure 2. Downregulation of PKC by PMA and OA. Quiescent RASMCs were incubated for 0, 3, 6, 12, and 24 hours at 37°C with 200 nmol/L PMA (top) and 100 µmol/L OA (bottom). Cells were harvested and protein-solubilized for Western blotting. Solubilized cellular proteins (10 µg) were resolved by SDS-PAGE on 7.5% polyacrylamide gels. After transfer to polyvinylidene membranes, PKC was detected with monoclonal antibodies to PKC, PKCµ, PKC, and PKC. Arrows indicate the position of molecular mass standards.

View larger version (23K): [in this window] [in a new window]   Figure 3. OA-stimulated [3H]thymidine uptake is blocked by downregulating PKC, whereas the response to 10% FBS is unaffected. The concentrations of OA indicated were added to quiescent RASMCs preincubated with 200 nmol/L PMA for 24 hours to deplete PKC. [3H]Thymidine uptake was measured 6 hours after it was added to the media. Data are presented as mean±SEM for three independent experiments in triplicate.

Effects of OA on Activation and Translocation of PKC in RASMCs
As shown in Table 2, treatment of intact RASMCs with 100 nmol/L PMA induced a typical translocation of PKC activity from the cytosol to membrane fraction. Treatment of intact RASMCs with OA from 12.5 to 200 µmol/L caused a significant concentration-dependent activation of PKC in the cytosol, which reached 328% of control at the 200 µmol/L concentration. However, OA did not significantly increase PKC activity in the membrane fraction. The OA-induced PKC activity was virtually abolished by preincubating the cells with 4 µmol/L bisindolylmaleimide I. Activation of cytosolic PKC was also eliminated by depleting the four isoforms of PKC detected in RASMCs using 24-hour incubation with 200 nmol/L PMA (Fig 4). The in vitro PKC activity assay (Table 3), performed by partially purifying PKCs from cell homogenates using DEAE-Sephacel chromatography, showed that OA induced a concentration-dependent increase in PKC activity that was similar in the presence or absence of Ca²⁺ (10 mmol/L EGTA), phosphatidylserine, and diolein. These data are consistent with evidence that OA directly and fully activates Ca²⁺-independent PKC isoforms, which constitute three of four isoforms detected in these primary RASMCs.^{13 14}

View this table: [in this window] [in a new window]   Table 2. Effects of OA and PMA on PKC Activity in RASMCs

View larger version (22K): [in this window] [in a new window]   Figure 4. The OA-induced increase in PKC activity was abolished by downregulating PKC with PMA. Quiescent RASMCs with/without PKC depletion were stimulated with 100 µmol/L OA for 5 minutes. Cytosol and particulate samples were prepared. PKC activity assay was carried out as described. Values represent the mean±SEM for three experiments in triplicate.

View this table: [in this window] [in a new window]   Table 3. In Vitro Stimulation of PKC Activity Assay by OA With and Without Ca2+

Effects of Various 18-Carbon NEFAs on [³H]Thymidine Uptake in RASMCs
Oleic (18:1 [cis]) and linoleic (18:2 [cis]) acids are potent activators of PKC, whereas stearic (18:0) and elaidic (18:1 [trans]) acids are not.^{13 14} Both oleic and linoleic acids induced significant concentration-dependent increases of thymidine uptake, whereas stearic and elaidic acids had very little effect. More specifically, at concentrations of 50, 100, and 200 µmol/L, OA led to 1.4-, 1.7-, and 1.8-fold increases, whereas comparable concentrations of linoleic acid were associated with 1.4-, 1.7-, and 2.0-fold increases, respectively (data not shown). In contrast, the 200 µmol/L concentrations of elaidic and stearic acids were associated with only 1.1- and 1.2-fold increases of thymidine uptake over control, respectively, in RASMCs, which were not statistically significant.

Effect of OA on MAP Kinase Activity
The MAP kinases are activated in response to PKC activation in vascular smooth muscle cells.²⁷ MAP kinase activity was measured by ³²P labeling of myelin basic protein in cytosolic extracts prepared from RASMCs under control conditions and after incubation with 100 µmol/L OA for 2, 5, 10, 30, and 60 minutes. The basal value for MAP kinase activity was 384±31 pmol/min per milligram of protein. As shown in Fig 5, OA (100 µmol/L) produced a time-dependent increase in MAP kinase activity that peaked at 10 minutes, reaching values 2.8 times greater than control (panel A). MAP kinase activity returned to basal values by 30 minutes. In RASMCs pretreated with PMA for 24 hours to deplete PKC, MAP kinase activity was greater than in untreated cells. However, OA no longer increased MAP kinase activity in the RASMCs depleted of PKC (panel B). The validity of the myelin basic protein phosphorylation assay as a measure of MAP kinase activity was confirmed by the ability of PMA to induce mobility shifts of immunoblotted MAP kinase isoforms²⁷ and PMA- and OA-induced tyrosine phosphorylation of immunoreactive MAP kinase (Fig 6). As shown, OA (100 µmol/L) and PMA (100 nmol/L) treatment induced a marked increase in tyrosine phosphorylation of immunoreactive MAP kinases in RASMCs, and these effects were blocked by PKC depletion. Densitometric scanning of the blots indicated that PMA caused a greater increase in tyrosine phosphorylation of MAP kinase than did OA (15.9 versus 7.3 times control, respectively).

View larger version (24K): [in this window] [in a new window]   Figure 5. Top, Time course of OA-stimulated MAP kinase activation in RASMCs is shown. Quiescent RASMCs were incubated with 100 µmol/L OA for the indicated times. The cells were harvested and prepared as described in "Materials and Methods" for MAP kinase activity assay. Values shown are the mean±SEM from three experiments. Bottom, PKC depletion inhibits OA-stimulated activation of MAP kinase in RASMCs. Quiescent RASMCs were pretreated for 24 hours with/without PMA and then stimulated with 100 µmol/L OA for 10 minutes as described in "Materials and Methods." Values shown are the mean±SEM from three experiments.

View larger version (16K): [in this window] [in a new window]   Figure 6. OA-induced tyrosine phosphorylation of MAP kinase was blocked by PKC depletion. Quiescent RASMCs were pretreated for 24 hours with/without PMA and then stimulated with 100 µmol/L OA or 100 nmol/L PMA for 10 minutes. Cell lysates were immunoprecipitated with antiphosphotyrosine, and then the immunoprecipitates were blotted with anti–MAP kinase antibody.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
There are two principal findings from the present study. First, physiological concentrations of OA induce proliferation of cultured RASMCs. Second, the mitogenic response of RASMCs to OA appears to be mediated by activation of PKC. These findings raise the possibility that the elevated nonesterified fatty acids, including OA, in obese hypertensive patients contribute to their excess vascular disease by activating PKC.²

Evidence That OA Induces Vascular Smooth Muscle Cell Proliferation
In the present study, the addition of OA to quiescent RASMCs was associated with a concentration-dependent increase in [³H]thymidine uptake, reaching values approximately double those of control (Fig 1). These data alone suggest that OA caused DNA synthesis in RASMCs. The observation that OA significantly increased cell number over 6 days to levels roughly double those of control cells indicates that this cis-unsaturated fatty acid has a mitogenic effect.

Evidence That OA Induces Mitogenesis in RASMCs by Activating PKC
Several observations in the present study suggest that OA causes vascular smooth muscle cells to proliferate by activating PKC. First, thymidine uptake, cell division, and activation of PKC in response to OA were all blocked by a selective PKC inhibitor, bisindolylmaleimide I. The selectivity of bisindolylmaleimide I for PKC compared with protein kinase A or Ca²⁺/calmodulin-dependent protein kinase is greater than the selectivity of other PKC inhibitors, such as staurosporine, polymyxin, or H-7.^{28 29 30} Bisindolylmaleimide I, in contrast to these other inhibitors, was not toxic to RASMCs under basal conditions, as assessed by thymidine, microscopy, and -actin staining. Unlike the other PKC inhibitors, bisindolylmaleimide I also did not significantly reduce thymidine uptake in response to 10% FBS. Similarly, epidermal growth factor induces the expression of primary response genes like c-fos in vascular smooth muscle cells by a PKC-independent pathway.^{31 32} Similarly, our data indicate that the mitogenic response to FBS, in contrast to OA, is not dependent on PKC activation. Bisindolylmaleimide I prevented the increase of thymidine uptake induced by 10 nmol/L PMA, which is a known PKC activator. These findings are all consistent with the concept that activation of PKC is responsible for OA-induced mitogenesis of RASMCs.

Second, depletion of PKC immunoreactivity by prolonged pretreatment of RASMCs with PMA prevented the concentration-dependent increase of thymidine uptake by RASMCs stimulated with OA. The depletion of PKC in the RASMCs after a 24-hour treatment with 200 nmol/L PMA was confirmed by immunoblots for the four PKC isoforms that we detected in these cells (, µ, , and ), including two atypical isoforms (Fig 2, top). Several reports indicate that the atypical isoforms ( and ) are not downregulated by phorbol esters.^{33 34} However, other laboratories have reported downregulation of PKC and other atypical PKC isoforms by prolonged incubation with PMA, despite the absence of a phorbol ester/diacylglycerol binding site.^{35 36 37 38} The reversal of OA's mitogenic effects by PMA pretreatment did not reflect nonspecific toxicity, since basal thymidine uptake and the thymidine response to 10% FBS were not reduced.

Third, treating intact RASMCs with OA induced a concentration-dependent increase of cytosolic PKC activity (Table 2), which resided predominantly in the Ca²⁺-independent fraction (Table 3). In attempting to explain how OA induced a sustained activation of PKC in cytosolic extracts prepared from the RASMCs (Table 2), it should be noted that most cells have fatty acid–binding proteins for transporting extracellular fatty acids intact to the cytosol.¹⁵ In preparing the cytosolic fraction from the RASMC lysate, some OA is probably retained and contributes to sustained activation of PKC. Since fatty acids activate PKC by binding to a site on the regulatory domain distinct from that for diolein/phorbol esters,^{39 40} an alternative explanation is that OA entering the cytosol before preparation of the lysate remains bound to PKC, leading to sustained activation. This PKC activation would be most apparent in the Ca²⁺-independent isoforms, which, in contrast to Ca²⁺-dependent PKCs, are more sensitive and fully activated by cis-unsaturated fatty acids, even in the absence of phosphatidylserine and diolein.^{13 14 39 40 41} Moreover, in the primary cultures of RASMCs examined in the present study, two of the three Ca²⁺-independent PKC isoforms identified are atypical ( and ) and do not bind diacylglycerol, which is lipid soluble, is restricted to the membrane, and is the major regulator of the classic Ca²⁺-dependent PKCs. Collectively, the observations suggest that OA activated PKC, which was a critical event in the mitogenic response. The data suggest, but do not prove, that activation of one or more of the Ca²⁺-independent PKC isoforms was primarily responsible for the mitogenic response of RASMCs to OA.

Fourth, OA activated MAP kinase in RASMCs (Figs 5 and 6). Activation of MAP kinase is a common and possibly critical event in the mitogenic response to several growth factors.^{27 42 43} Activation of MAP kinase by OA was inhibited by downregulating PKC with PMA. These data suggest that OA activated PKC with subsequent activation of MAP kinase. The results of the present study coincide with reports indicating that activation of PKC, in this case by cis-unsaturated OA,^{13 14 39 40 41 44 45} can lead to a mitogenic response in vascular smooth muscle cells.^{46 47}

Fifth, the capacity of nonesterified fatty acids to activate PKC varies markedly. Longer-chain cis-unsaturated fatty acids such as oleic (18:1 [cis]) and linoleic (18:2 [cis]) acids are potent activators of PKC, whereas stearic (18:0) and elaidic (18:1 [trans]) acids are much less potent activators of PKC.^{39 40 44 45} Stimulation of thymidine uptake by these four fatty acids paralleled their known capacity to activate PKC. More specifically, oleic and linoleic acids, at 50 to 200 µmol/L, induced a concentration-dependent increase of thymidine uptake in RASMCs. In contrast, identical concentrations of stearic and elaidic acids did not cause a significant enhancement of thymidine uptake. The variable mitogenic responses to the four different 18-carbon fatty acids also provide indirect evidence against a nonspecific nutritive effect on cell growth.

One potential caveat to interpretation of the linoleic acid–induced mitogenic effect is the multiple eicosanoid products generated from this fatty acid, which can participate in the growth response.⁴⁸ In contrast to linoleic acid, OA is not a usual substrate for the cyclooxygenase, lipoxygenase, or epoxygenase pathways. Therefore, although activation of PKC is crucial in the mitogenic response to OA, this may not be true for linoleic acid,⁴⁸ despite the similar degree of thymidine uptake induced by these two fatty acids.

Limitations and Alternative or Adjunctive Explanations
Although activation of PKC can change membrane ion transport,⁴⁹ nonesterified fatty acids also directly modulate multiple membrane transport processes, including Na⁺-H⁺ antiport and Ca²⁺ flux,⁵⁰ which may influence growth responses. The capacity of PKC inhibition with bisindolylmaleimide I and downregulation of PKC with PMA to completely prevent the effects of OA on thymidine uptake makes it unlikely that the mitogenic response was mediated by primary effects of fatty acids on membrane transport. However, changes in ion transport secondary to activation of PKC by fatty acids⁴⁹ may have participated in cell growth.

Potential Implications for Vascular Disease in Patients With Insulin Resistance
Insulin resistance emerges as a potential common pathogenetic denominator in cardiovascular risk factor clustering and excess cardiovascular morbidity and mortality.^{1 2 3 4 5 6} The mechanisms linking abnormalities of insulin dynamics and action to cardiovascular risk and events remain incompletely defined. Patient groups with resistance to insulin-mediated glucose disposal, such as those with abdominal obesity,¹⁰ diabetes,⁹ obesity hypertension,⁸ and renal failure,^{51 52} also have resistance to insulin's capacity to lower plasma nonesterified fatty acids. Resistance to insulin's fatty acid actions in clamp studies corresponds to elevation in circulating plasma fatty acids over 24 hours at other times during the day.^{11 12} The results of the present study raise the possibility that elevations in plasma nonesterified fatty acids, particularly OA, may contribute to the excess vascular disease observed in patients with insulin resistance^{1 5} by activating PKC and contributing to vascular smooth muscle cell proliferation.

	Selected Abbreviations and Acronyms

 

MAP = mitogen-activated protein NEFA = nonesterified fatty acid PKC = protein kinase C PMA = phorbol 12-myristate 13-acetate RASMC = rat aortic smooth muscle cell

	Acknowledgments

 
This study was supported by National Institutes of Health grants HL-43164 (Dr Egan) and CA-58640 (Dr Meier) and by the Medical University of South Carolina Institutional Research Fund (Dr Morinelli).

Received January 25, 1996; accepted June 10, 1996.

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