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Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:445-453

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:445-453.)
© 1997 American Heart Association, Inc.

Articles

Oxidized LDL Mediates the Release of Fibroblast Growth Factor-1

Natalya M. Ananyeva; Alexey V. Tjurmin; Judith A. Berliner; Guy M. Chisolm; Gene Liau; Jeffrey A. Winkles; ; Christian C. Haudenschild

From the Departments of Experimental Pathology (N.M.A., A.V.T., C.C.H.) and Molecular Biology (G.L., J.A.W.), Holland Laboratory, American Red Cross, Rockville, Md; the Department of Pathology and Medicine/Cardiology, University of California at Los Angeles (J.A.B.); and the Department of Cell Biology, The Cleveland (Ohio) Clinic Foundation (G.M.C.).

Correspondence to C.C. Haudenschild, Department of Experimental Pathology, Holland Laboratory, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855. E-mail haudenschildc{at}usa.redcross.org.


*    Abstract
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Abstract Fibroblast growth factor-1 (FGF-1) and lipoproteins play an important role in atherogenesis. In the present study, we explored a possible mechanism by which abnormal lipid metabolism could be linked to the proliferative aspects of the disease. We tested oxidized LDL (oxLDL) as a possible pathophysiological mediator of the release of FGF-1, using FGF-1–transfected mouse NIH 3T3 cells and FGF-1–transfected rabbit smooth muscle cells, and compared it with the release caused by elevated temperature. Immunoblot analysis showed that oxLDL induced the release of FGF-1 in a concentration-dependent manner from 10 to 100 µg/mL. The effect correlated with the extent of oxidative modification of LDL and was maximal within 4 hours of exposure of cells to oxLDL. In contrast to the temperature stress–induced FGF-1 secretion pathway, FGF-1 released in response to oxLDL (1) appeared in the conditioned medium as a monomer, (2) appeared independently of the presence of either actinomycin D or cycloheximide, and (3) was neither enhanced nor inhibited by brefeldin A. We did not detect cell loss, significant morphological changes, changes in growth characteristics, or other indications of lethal toxicity in oxLDL-treated cells. Although the level of lactate dehydrogenase activity was elevated after oxLDL exposure, the calculations showed that >90% of the FGF-1 was released by viable cells. We propose that oxLDL-induced FGF-1 release is mediated by sublethal and apparently transient changes in cell membrane permeability. In the environment of an atherosclerotic lesion, oxLDL-induced FGF-1 release may be among the mediators of endothelial and smooth muscle cell proliferation.


Key Words: fibroblast growth factor-1 • oxidized LDL • atherosclerosis • sublethal injury


*    Introduction
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 *Introduction
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 down arrowResults
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Fibroblast growth factor-1 belongs to a family of polypeptides currently consisting of 10 structurally related members with a broad biological base. FGF-1 and FGF-2 induce the proliferation and differentiation of a wide range of cells of mesodermal and neuroectodermal origin and are recognized as potent chemoattractive and angiogenic factors.1 2 3 4 Experimental evidence supports an important role of the FGFs in development, wound healing, and a variety of pathophysiological situations, including atherosclerosis. Experiments on animals demonstrated that these growth factors can stimulate reendothelialization, mediate proliferation of SMCs, and induce adventitial angiogenesis in balloon-injured carotid arteries.5 6 7 8 9 10 Injury of rat arteries leads to an increase in FGF receptors in vascular SMCs, further suggesting involvement of these growth factors in the genesis of intimal hyperplasia.11 The FGFs are found in macrophages, SMCs, and ECs, the principal cell types involved in atherosclerosis, with FGF-1 expression in macrophages predominating.12 13 Studies on human tissues demonstrate patterns of FGF distribution in atheroma different from those in normal vessels.12 13 Both FGF-1 and FGF-2 and their receptors can be detected in the normal vessel wall. Atherosclerotic lesions are characterized by a lower level of FGF-2 expression, whereas FGF-1 expression increases, predominantly in the areas of vascularization.13

Unlike the majority of known growth factors, both prototype members of the FGF family, FGF-1 and FGF-2, lack a classic signal sequence for secretion. Although an intracellular function for these factors cannot be completely ruled out, it is known that both factors have to be externalized to interact with specific high-affinity receptors and to induce a mitogenic signal. So far, no general concept explaining the release of FGF prototypes from intracellular sources has been accepted, but several mechanisms have been proposed. According to one hypothesis, such a mechanism could be active transport of FGF directly through the plasma membrane by an unknown alternative secretion pathway.14 Indeed, active secretion of FGF-1 from FGF-1–transfected mouse NIH 3T3 fibroblasts can be induced by temperature stress.15 Alternatively, FGF could be released as a consequence of cell damage, such as transient membrane disruptions16 17 18 19 20 21 22 or cell death.18 23 24 It is also possible that exocytosis may be one of the pathways used by FGF-2.25 Our hypothesis is that in the pathological environment of an atherosclerotic lesion, one of the principal agents capable of releasing FGF-1 could be oxLDL.

Retention of LDL in the vessel wall with subsequent oxidation now is considered to be a key event in the early progress of atherosclerotic lesions.26 27 28 29 Exerting various effects on artery wall cells, oxLDL is involved in different stages of the atherosclerotic process, including fatty streak formation, formation of the definitive plaque, and thrombus formation.30 31 By altering the motility of the principal cell types involved in angiogenesis, oxLDL promotes recruitment and retention of monocyte/macrophages during atherosclerosis.32 33 34 35 36 37 Products of LDL oxidation are accumulated by both macrophages and SMCs, which transforms them into foam cells.38 OxLDL can act as the activator of the main cell types by increasing production of different growth factors and cytokines.39 40 41 OxLDL may also be responsible for cell injury observed in atherosclerosis. The toxicity of oxLDL was demonstrated with cultured vascular SMCs, ECs, and fibroblasts, proliferating cells being most vulnerable.42 43 44 45 46 At subtoxic concentrations, oxLDL has been shown to have a proliferative effect on vascular SMCs47 48 and macrophages.49

In this study, we explored a possible mechanism by which modified lipoproteins could be linked to the pro- liferative aspects of atherosclerosis. We tested oxLDL as a possible pathophysiological mediator of the release of FGF-1 and compared it with the release caused by elevated temperature.15 50 We show here that oxLDL but not native LDL is capable of inducing the release of FGF-1 from cultured fibroblasts and SMCs. Release of FGF-1 from these cells does not involve cell death but rather is due to transient and reversible changes in cell membrane permeability of viable cells. These results provide a plausible mechanism for the release of FGFs during atherosclerotic lesion progression.


*    Methods
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Fibroblast Cell Culture
The experiments were performed on murine NIH 3T3 cells transfected with the eukaryotic expression vector pMEXneo51 containing a synthetic gene encoding FGF-1 (residues 21 to 154).52 The functional and biological features of FGF-121-154 protein are indistinguishable from those of the full-length protein.53 The cells were grown to confluence in fibronectin-covered (Sigma) 100-mm culture dishes in DMEM (Gibco) containing 10% (vol/vol) bovine calf serum (HyClone) and Geneticin (400 µg/mL, Sigma) at 37°C in a humidified atmosphere of 92% air/8% CO2. Before the experiment, the cells were incubated for 16 hours in endothelial SFM (Gibco BRL). At time zero, the medium was exchanged for a fresh portion containing 10 U/mL heparin (5 mL/dish), and the cells were exposed to elevated temperature (42°C) for 2 hours15 or maintained at 37°C for the indicated times in the presence of various concentrations of native or oxidized LDL. In some experiments, the cells were preincubated with either actinomycin D (5 µg/mL, Sigma) or cycloheximide (5 µg/mL, Sigma) for 2 hours or brefeldin A (0.5 µg/mL, Sigma) for 30 minutes.

SMC Culture
Primary cultures of SMCs were isolated from the aorta of New Zealand White rabbits by enzymatic digestion with collagenase and elastase as previously described.54 They were routinely cultured in medium 199 (Gibco) with 10% FBS (HyClone), 4 mmol L-glutamine, 100 U/mL penicillin G sodium, 100 µg/mL streptomycin sulfate, and 0.25 µg/mL amphotericin B. SMCs were seeded at a density of 5x103 cells/cm2 and cotransfected (10:1 ratio) with the plasmid p267-3 containing human FGF-121-154 cDNA55 and pSV2 neo56 by a modified CaPO4 transfection protocol (Stratagene). A number of Geneticin-resistant cell lines expressing various levels of FGF-121-154 were generated, and detailed analysis of these cell lines will be described elsewhere. Several cell lines constitutively expressing a high level of FGF-121-154 were used for the studies described here. The transfected cells were grown to confluence in DMEM (Gibco) containing 10% (vol/vol) FBS and Geneticin (400 µg/mL) at 37°C in a humidified atmosphere of 92% air/8% CO2. The experiments were performed exactly as described for FGF-1–transfected NIH 3T3 cells.

LDL Isolation and Oxidation
LDL from pooled human sera was isolated by ultracentrifugation,57 dialyzed against 150 mmol/L NaCl containing 0.3 mmol/L EDTA, sterilized with 0.2-µm Millipore membranes, and stored at 4°C under nitrogen. LDL was oxidized to various extents. For mildly modified LDL, LDL was oxidized by mild iron oxidation.58 More severely oxidized LDL was obtained by dialysis in 0.15 mol/L NaCl, pH 6.5, containing 6 µmol/L CuSO4 for 24 to 48 hours. After oxidation, the LDL was further dialyzed in PBS, pH 7.4, containing 0.01% EDTA. All active preparations contained 2 to 6 nmol/L thiobarbituric acid–reactive substances/mg cholesterol. The electrophoretic mobility of oxLDL relative to that of native LDL was 2.8±0.2; mildly oxidized LDL had the same electrophoretic mobility as native LDL. All lipoproteins were stored in PBS containing EDTA (0.3 mmol/L) and BHT (0.1 mmol/L). Since levels >150 µg protein/mL oxLDL have been reported to be cytotoxic,43 44 59 60 61 we tested the range of 10 to 100 µg/mL and typically used 50 µg/mL of oxLDL.

Immunoblot Analysis
Conditioned media collected from control, heat-treated, or LDL-treated NIH 3T3 cells or rabbit SMCs were centrifuged at 600g for 10 minutes, and 100-µL aliquots were taken for LDH assessment. Thereafter, the proteinase inhibitors PMSF, leupeptin, and aprotinin were added to final concentrations of 1 mmol/L, 1 µg/mL, and 1 µg/mL, respectively, and 4.5 mL of the medium was mixed with 200 µL of heparin-Sepharose (Pharmacia) equilibrated in 50 mmol/L Tris-HCl, pH 7.5, and 10 mmol/L EDTA. In experiments requiring reducing conditions, the conditioned media were incubated with DTT (final concentration, 1 mmol/L) for 2 hours at 37°C before exposure to heparin-Sepharose. Binding of FGF-1 proceeded overnight at 4°C with rotation; then the supernatants were discarded, the pellets were washed twice with 1 mL 50 mmol/L Tris-HCl, pH 7.5, 1 mmol/L EDTA, and 0.15 mol/L NaCl, and the bound protein was eluted with 100 µL of either reducing or nonreducing sample buffer for SDS-PAGE.

The samples representing the equal numbers of cells for different experimental conditions were resolved by SDS-PAGE (15% wt/vol acrylamide) and transferred to nitrocellulose filters. The filters were incubated in blocking solution containing 5% wt/vol dry milk in TBS-Tw (50 mmol/L Tris-HCl, pH 7.5, 0.15 mol/L NaCl, 3 mmol/L KCl, and 0.1% vol/vol Tween 20) for 1.5 hours at 37°C and incubated with a rabbit anti-human FGF-1 antibody (1 µg/mL in TBS-Tw) overnight at 4°C. After three washes in TBS-Tw, the membranes were incubated with a goat anti-rabbit antibody conjugated with horseradish peroxidase (Sigma) for 1.5 hours at room temperature, and the proteins were detected with ECL detecting reagents (Amersham). Recombinant human FGF-121-154 served as a positive control. The same recombinant protein was used to immunize rabbits for antibody generation. Molecular weight markers were purchased from Bio-Rad.

Cell Viability Assays
1. The cytotoxicity of oxLDL and the loss of individual cells were assessed by the trypan blue exclusion test. At the end of incubation, the cells were washed once with PBS and then stained for 5 minutes with 0.1% (wt/vol) trypan blue dissolved in PBS. The monolayers were then washed twice with PBS, and the numbers of nonviable cells (ie, those cells that failed to exclude the dye) were counted in 10 fields (d=1.25 mm) per dish. The total number of cells in each dish was counted three times in a Coulter counter after the cells were detached by treatment with 0.125% (wt/vol) trypsin-Versene solution (Gibco BRL).

2. After LDL treatment, cells were replated onto 24-well plates with a density of 2x102 cells/mm2. At the indicated time points, triplicate wells were trypsinized, and the cells were counted in a Coulter counter.

3. Morphological changes in control and oxLDL-treated cells were assessed by electron microscopy. Conditioned media were collected, and the cells were fixed directly in the dishes in 2.5% glutaraldehyde in PBS for 2 hours at 4°C. The cells were then washed in PBS at 4°C, postfixed in 1% OsO4 for 1 hour, washed in distilled water, dehydrated, and embedded in block molds in 100% epoxy resin. Sections (500 to 700 nm) were stained with uranyl acetate and lead citrate and observed in the CM12 transmission electron microscope (Phillips).

4. LDH activity in the conditioned media collected from {approx}107 control, heat-shocked, and LDL-treated cells was assessed by spectrophotometric enzymatic analysis (340 nm) according to the manufacturer's instructions (Sigma).

5. The viability of cells after a 4-hour incubation with oxLDL was assessed by a colorimetric assay (based on MTT labeling reagent according to the Cell Proliferation Kit I, Boehringer Mannheim). The cell metabolic activity was determined from absorbance (A550 nm to A690 nm) of the formazan product in oxLDL-treated cells compared with that in control cells and expressed as percentage of control.

6. The permeability of cell membranes to a low-molecular-weight dye, PI (MW=668), was assessed after a 4-hour incubation with oxLDL or control medium. The medium was changed to a fresh portion of SFM containing 100 µg/mL PI. After a 30-minute incubation at 37°C, the cells were washed twice with SFM, formalin-fixed for 10 minutes, and analyzed in a fluorescence microscope. The number of PI-positive cells and the total cell number were counted in 10 microscopic fields per dish, and the average percentage of PI-positive cells was calculated. The experiment was repeated three times.

Mathematical Evaluation
Quantitative densitometry of ECL-developed films was performed with a ScanJet II CX/T scanner (Hewlett Packard) by measurement of the integral densities of experimental and standard bands. Different amounts of recombinant FGF-121-154 were used to construct a calibration curve, which represented an asymptotic regression with initial linear stretch and reaching a saturation plateau at high quantities of FGF-1. Quantitative measurements of experimental bands were performed only within linear and initial nonlinear areas of the calibration curve.

All data are presented as mean±SD. Comparison of the data was performed with Student's t test and was considered significantly different if P<.05.


*    Results
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 up arrowMethods
 *Results
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oxLDL Induces FGF-1 Release From Fibroblasts
FGF-121-154–transfected NIH 3T3 cells represent a well-characterized model to study FGF-1 release from intact cells. We used these cells to study the effect of exposure to native LDL and LDL oxidized to two different extents. FGF-1 was detectable in the supernatants of heat-treated cells (Fig 1Down, lane 4), confirming earlier findings.15 The effect of LDL on FGF-1 release correlated positively with the degree of oxidation: the conditioned medium from untreated cells (Fig 1Down, lane 3) and cells treated with 100 µg/mL native LDL (Fig 1Down, lane 5) contained no FGF-1, whereas the same concentration of highly oxidized LDL induced FGF-1 release from transfected cells after a 2-hour incubation at 37°C (Fig 1Down, lane 7). It is noteworthy that oxLDL under the indicated conditions induced a fourfold higher level of FGF-1 than temperature stress (Fig 1Down, lanes 4 and 7). Mildly oxidized LDL exerted an intermediate effect (Fig 1Down, lane 6). A combination of temperature stress and oxLDL had a synergistic effect (2 to 4 times more than the expected additive amount) on the release of FGF-1 (Fig 1Down, lanes 9 to 11).



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  		Figure 1. Release of FGF-1 from NIH 3T3 cell pMEXneo/FGF-121-154 in response to temperature and/or LDL after variable oxidation. Conditioned media were treated with 1 mmol/L DTT and adsorbed to heparin-Sepharose, and eluates representing same number of cells (typically 107) were analyzed by immunoblot analysis for FGF-1. Lanes 1 and 2 show recombinant FGF-121-154 (80 and 25 ng); lane 3, control: no LDL, 37°C, 2 hours; lane 4, heat shock: no LDL, 42°C, 2 hours; lanes 5 to 7, conditioned media from cells maintained in presence of 100 µg/mL native, mildly oxidized, and highly oxidized LDL at 37°C, 2 hours; lane 8, MW markers; lanes 9 to 11, conditioned media from cells maintained in presence of 100 µg/mL native, mildly oxidized, and highly oxidized LDL at 42°C, 2 hours; lane 12, preexperimental conditioned medium (SFM, 16 hours); and lane 13, cell lysate (equivalent to 106 cells). SDS-PAGE: 15% acrylamide, reducing conditions. Immunoblot is representative of 12 independent experiments.

The effect of oxLDL was time- and concentration-dependent: FGF-1 was released at a concentration of oxLDL as low as 10 µg/mL and increased as the oxLDL concentration was increased up to 100 µg/mL (Figs 1Up and 2Down). The indicated concentration range was chosen to avoid the reported cytotoxic effect of oxLDL at concentrations >150 to 200 µg/mL.43 44 59 60 61



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  		Figure 2. Dependence of FGF-1 release on concentration of oxLDL and on time of incubation. Lane 1 shows MW markers; lanes 2, 3, and 14, recombinant FGF-121-154 (160, 80, and 40 ng); lanes 4 and 9, conditioned medium from control cells (2 vs 4 hours); lanes 5 and 10, conditioned medium from heat-shocked cells (2 vs 4 hours); lanes 6 to 8, conditioned media from cells maintained in presence of 50, 25, and 10 µg/mL oxLDL, respectively, at 37°C for 2 hours; and lanes 11 to 13, oxLDL (50, 25, and 10 µg/mL), 37°C, 4-hour incubation. SDS-PAGE: 15% acrylamide, reducing conditions. Immunoblot is representative of 4 independent experiments.

The kinetics of FGF-1 release in response to oxLDL are shown in Fig 3Down. Significant levels of extracellular FGF-1 were detected after 2 hours of exposure of NIH 3T3 cells to oxLDL. The maximal release was observed after a 4-hour incubation, whereas the FGF-1 content in the conditioned medium did not increase further with time (up to 16 hours).



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  		Figure 3. Time course of FGF-1 release in response to oxLDL. Cells were incubated in SFM (control) or in SFM in presence of 50 µg/mL oxLDL for indicated periods of time. Thereafter, conditioned media were analyzed by immunoblot assay, and quantities of FGF-1 released were measured densitometrically as described in "Methods." Solid line indicates oxLDL treatment; dotted line, control. Each point represents average of three determinations. Amounts of FGF-1 are normalized per 107 cells.

OxLDL-Released FGF-1 Appears as a Monomer
Elevated temperature releases FGF-1 in the form of a biologically inactive homodimer with a low heparin affinity,15 and pretreatment with a reducing agent is necessary to restore its heparin-binding ability. We examined whether oxLDL-released FGF-1 also exists as a dimer. FGF-1 in the conditioned media of fibroblasts treated by heat or oxLDL was affinity-selected on heparin-Sepharose without pretreatment with DTT and subjected to SDS-PAGE under nonreducing conditions. Western blot analysis demonstrated that under these conditions, heat-released FGF-1 was undetectable (Fig 4Down, lane 3). In contrast, FGF-1 in the conditioned medium of fibroblasts incubated with oxLDL bound to heparin-Sepharose in the absence of DTT, indicating that oxLDL releases the monomeric form of FGF-1 (Fig 4Down, lane 4).



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  		Figure 4. Comparison of temperature stress–induced and oxLDL-induced FGF-1 release in nonreducing conditions. Conditioned media from NIH 3T3/FGF-121-154 transfectants were collected after temperature stress (42°C, 2 hours) or after 4-hour incubation with 50 µg/mL oxLDL and processed as described in "Methods." Media were adsorbed with heparin-Sepharose without pretreatment with DTT, and eluates wereanalyzed by immunoblot analysis for FGF-1. Lane 1 shows MW markers; lane 2, recombinant FGF-121-154 (100 ng); lane 3, heat shock: no LDL, 42°C, 2 hours; and lane 4, oxLDL, 50 µg/mL, 37°C, 4 hours. SDS-PAGE: 15% acrylamide, nonreducing conditions. Immunoblot is representative of 12 independent experiments.

OxLDL-Mediated Release of FGF-1 Is Independent of De Novo Synthesis and the ER Golgi Apparatus
We used the drugs actinomycin D (an RNA synthesis inhibitor) and cycloheximide (a protein synthesis inhibitor) to examine whether the release of FGF-1 by oxLDL involves transcription or translation. FGF-1 was accumulated in conditioned medium independently of the presence of either actinomycin D (Fig 5Down, lane 6) or cycloheximide (Fig 5Down, lane 8), indicating that the growth factor is released from the presynthesized cytoplasmic pool. In this respect, oxLDL-induced release of FGF-1 differs from temperature stress–induced secretion of the growth factor, which can be effectively suppressed by these inhibitors.15



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  		Figure 5. OxLDL-induced FGF-1 release in presence of actinomycin D, cycloheximide, or brefeldin A. Confluent NIH 3T3 pMEXneo:FGF-1 transfectants were pretreated with actinomycin D (5 µg/mL) or cycloheximide (5 µg/mL) for 2 hours or with brefeldin A (0.5 µg/mL) for 30 minutes. After pretreatment, media were exchanged for SFM containing 50 µg/mL oxLDL/5 µg/mL actinomycin D, 50 µg/mL oxLDL/5 µg/mL cycloheximide, or 50 µg/mL oxLDL/0.5 µg/mL brefeldin A, and monolayer was maintained at 37°C for 4 hours. Control cells were maintained in SFM and in SFM containing 50 µg/mL oxLDL. Media were adsorbed with heparin-Sepharose without pretreatment with DTT, and eluates representing equal number of cells were analyzed by immunoblot analysis for FGF-1. Lane 1 shows control; lane 2, MW markers; lane 3, oxLDL, 50 µg/mL, 37°C, 4 hours, 1/2 of sample volume loaded; lanes 4 and 5, recombinant FGF-121-154 (100 and 50 ng); lane 6, oxLDL in presence of actinomycin D; lane 7, actinomycin D; lane 8, oxLDL in presence of cycloheximide; lane 9, cycloheximide; lane 10, oxLDL in presence of brefeldin A; and lane 11, brefeldin A. SDS-PAGE: 15% acrylamide, nonreducing conditions. Immunoblot is representative of 5 independent experiments.

We also tested the effect of brefeldin A, a pharmacological agent known to impair the secretory function of the ER Golgi apparatus,62 on FGF-1 release in response to oxLDL. The presence of brefeldin A (0.5 µg/mL) neither inhibited nor significantly enhanced the FGF-1 level in the conditioned medium (Fig 5Up, lane 10). Interestingly, in the previous heat-shock experiments, this agent caused a paradoxical enhancement of the temperature-induced release of FGF-1.50 These results suggest that the release of FGF-1 in response to oxLDL follows a pathway different from both the conventional secretory pathway mediated by the ER Golgi apparatus and from the temperature-induced release pathway.

OxLDL-Induced FGF-1 Release Is Not a Result of Cell Death
Since oxLDL-induced FGF-1 release most likely has a mechanism different from that of temperature-induced secretion of the factor, we asked whether the accumulation of FGF-1 in the medium after exposure to oxLDL was a result of cytotoxicity of oxLDL. Products of LDL oxidation at concentrations of >=150 µg/mL are known to be cytotoxic to many cell types, including ECs, SMCs, and fibroblasts.43 44 59 60 61 We used lower concentrations (10 to 100 µg/mL) and did not observe toxicity with multiple methods.

  1. After a 4-hour incubation with 100 µg/mL oxLDL (maximal time and concentration), no morphological evidence of cell damage, shape changes, or cell detachment was seen. We did not detect a substantial cell loss: <0.3% of the cells in the worst case and 0.05±0.02% on average (obtained from 35 calculations) were positive in the trypan blue exclusion test, compared with 0.02±0.01% in control cells.
  2. Replating of cells after a 4-hour incubation in the absence or presence of oxLDL yielded similar plating efficiencies and cell growth curves (Fig 6Down).

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              		Figure 6. Replating of cells after exposure to oxLDL. Control cells and cells after 4 hours of treatment with oxLDL (100 µg/mL) were plated onto 24-well plates with a density of 2x102 cells/mm2. At indicated time points, triplicate wells were trypsinized, and cells were counted in a Coulter counter.

  3. More than 98% of oxLDL-treated cells remained metabolically active, as evidenced by MTT-based colorimetric quantification of viable cells.
  4. Electron microscopy did not reveal any disturbances or rearrangements in Golgi apparatus, ER, or other organelles in oxLDL-treated cells. The cell and nuclear membranes appeared intact (Fig 7Down bottom and top, respectively).

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              		Figure 7. Electron microscopy of FGF-1–transfected NIH 3T3 fibroblasts after 4 hours of exposure to 100 µg/mL oxLDL. Magnification x45 000.

OxLDL-Induced FGF-1 Is Released Via Transient Changes in Cell Membrane Permeability
The above data argue strongly against cell lysis induced by oxLDL as a reason for FGF-1 release and thus indicate that FGF-1 is released by the effects of oxLDL on viable cells. However, when we plotted the level of LDH activity measured in the conditioned medium of oxLDL-treated cells against the amount of FGF-1 released in different experiments or in different conditions (oxLDL dose and time of exposure), we found a strong correlation between these two parameters (Fig 8Down). LDH is a cytosolic protein freely diffusible from damaged cells. To determine whether the LDH and FGF-1 in the conditioned medium came from a few lysed cells or from many viable but transiently leaky cells, we compared the amounts of LDH and FGF-1 released with the maximum amounts of each that could theoretically be released from all accounted dead cells for each experimental condition. To estimate the total available amounts of LDH and FGF-1, cells were lysed intentionally by use of hypotonic shock: 5.5 U LDH and 1500 ng FGF-1 were released per 107 cells (the average values of three determinations). In the worst case, according to the trypan blue exclusion test, 0.3% of the cells (3x104 per 107 cells) were lysed by oxLDL, which would release 16.5 mU LDH and 4.5 ng FGF. The average number of trypan blue–positive cells was lower (0.05±0.02%), accounting for accumulation of 2.75 mU LDH and 0.75 ng FGF-1. The experimental values for FGF-1 release by 50 µg/mL oxLDL within 4 hours constituted 360±265 mU/107 cells LDH and 130±105 ng/107 cells FGF-1 (the average values of seven determinations). This suggests that lysed cells could account for only a minor portion, <5%, of LDH and FGF-1 measured; the major portion appeared to have been released by viable cells, perhaps through temporary leaks in cell membranes created by oxLDL. From the plot in Fig 8Down, we calculated the average LDH/FGF ratio in medium after exposure to oxLDL to be 2.3:1 (mU/ng). This was almost twice as low as that in the cell lysate (3.7:1 mU/ng). This would be consistent with the assertion of leaking cell membranes, because the smaller molecule (FGF-1, MW=17 kD) would more easily escape the cell than the larger one (LDH, MW=140 kD).



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  		Figure 8. Correlation between FGF-1 release and LDH activity. For each experimental condition, LDH activity was measured in conditioned medium, and amount of FGF-1 released was determined from immunoblots densitometrically as described in "Methods" and normalized per 107 cells. In total, 35 experimental points representing different experiments or different conditions (oxLDL dose and time of exposure) are included in plot.

Postulating that nonlethal and possibly transient and short-lived changes in cell membrane permeability due to sublethal toxicity of oxLDL may be a mechanism for FGF-1 release, we expected that after exposure to oxLDL, the cells would be permeable to a low-molecular-weight dye, PI (MW=668 D). The results of the experiment showed that PI-positive cells constituted {approx}7.2±3.4%, compared with 0.01±0.02% in control cells (the average values from three experiments). This was consistent with the amount of FGF-1 released after exposure to oxLDL (130±105 ng/107 cells in releasate versus 1500 ng/107 cells in lysate, or 8% to 10% of the total intracellular pool of FGF-1). The PI-positive cells had normal microscopic appearance and were trypan blue–negative. These data support our hypothesis that oxLDL imposes nonlethal changes in cell membrane permeability that result in FGF-1 release into the medium.

Oxidized LDL Can Also Promote FGF-1 Release From Vascular SMCs
We next examined whether a similar release of FGF-1 can be induced by oxLDL treatment of cultured SMCs. Although SMCs express FGF-1, the level of expression is not sufficient to allow detection of the factor in conditioned medium. To overcome this difficulty, we transfected rabbit SMCs with an FGF-1–containing vector. Several cell lines expressing abundant levels of FGF-1 were isolated and used to study the effect of native LDL, oxLDL, or heat treatment. The effect on one representative cell line analyzed in nonreducing conditions is shown in Fig 9Down. The FGF-1 level in conditioned medium from the cells treated with native LDL did not differ from that of control cells (Fig 9Down, lanes 2 and 3). However, both heat-shock and oxLDL induced the release of FGF-1 (Fig 9Down, lanes 4 and 5). Interestingly, the temperature stress–induced FGF-1 was released in both a dimeric form and a monomeric form (Fig 9Down, lane 4). The amount of temperature-induced FGF-1 was higher than previously reported for FGF-1–transfected NIH 3T3 fibroblasts. In contrast, oxLDL-mediated FGF-1 was released as a slightly larger monomer (Fig 9Down, lane 5). As with fibroblasts, the level of FGF-1 released by oxLDL was considerably higher than that observed for heat-shocked cells. As for the 3T3 cells, the effect of oxLDL on SMCs was also not due to cell death (data not shown). We conclude that oxLDL induces the release of FGF-1 from SMCs via a mechanism similar to that demonstrated for fibroblasts.



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  		Figure 9. Release of FGF-1 from FGF-1–transfected rabbit SMCs in response to temperature or native or highly oxidized LDL. Experiment was performed as described for NIH 3T3 fibroblasts in `'Methods." Lane 1 shows MW markers; lane 2, control, 37°C, 4 hours; lane 3, native LDL, 50 µg/mL, 37°C, 4 hours;lane 4, heat-shock: 42°C, 2 hours; and lane 5, oxLDL, 50 µg/mL, 37°C, 4 hours. Immunoblot represents one of 4 independent experiments.


*    Discussion
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 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
FGF-1 does not contain a signal sequence and is not secreted via the classic ER Golgi pathway. Therefore, it is likely that there can be alternative mechanisms for its exit into the extracellular compartment to exert its biological function. Secretion of FGF-121-154 from the FGF-1–transfected NIH 3T3 fibroblasts in response to temperature stress15 is consistent with the in vivo situation in which FGF-1 expression is exaggerated in tissues undergoing inflammation.63 In the environment of the atherosclerotic lesion, however, factors other than temperature stress, such as oxLDL, would be expected to be more relevant.

As an experimental model to study the effect of oxLDL on FGF-1 release, we used FGF-121-154–transfected NIH 3T3 fibroblasts producing FGF-1 in great abundance (1.5 µg/107 cells), which significantly facilitated experimentation. OxLDL at concentrations >150 to 200 µg/mL is cytotoxic to cultured SMCs, ECs, and fibroblasts.43 44 59 60 61 The highest vulnerability is observed in rapidly proliferating cells, whereas quiescent cells are less susceptible to the cytotoxic effect of oxLDL.43 46 Therefore, we experimented on confluent cell cultures and focused on studying the effects of nontoxic concentrations of oxLDL, which are likely to be more relevant for the in vivo situation. We found that oxLDL induced the release of FGF-1 from FGF-1–transfected fibroblasts but native LDL did not. The response was proportional to the degree of oxidation of LDL. Thus, oxidation appeared to be essential for the ability of LDL to induce the release of FGF-1. Similar results were obtained in the analogous experiments using FGF-1–transfected rabbit SMCs, which are more relevant to the in vivo situation in the arterial wall. Taken together, these data indicate that oxLDL induction of FGF-1 release is not fibroblast-specific. In the milieu of the atherosclerotic lesion, oxLDL could promote FGF-1 release from various FGF-1–containing cell types, potentially including macrophages, ECs, and SMCs.

The exact levels of oxLDL in the atherosclerotic lesion are difficult to measure and can be different in different locations. Although local cytotoxic levels of oxLDL may account for cell necrosis in the lesions, oxLDL gradients can reasonably be expected in these heterogeneous plaque tissues, which include the range of concentrations used here, explaining one mechanism by which the pathological lipoprotein could also be responsible for cell proliferation.

Although both oxLDL and elevated temperature induce FGF-1 release, our results suggest that different regulatory mechanisms are involved in these processes. FGF-1 released by oxLDL exposure appears directly as a monomer, ie, as it is in the cell cytoplasm. In contrast, after temperature-induced secretion, extracellular FGF-1 is found in a dimeric form that is not able to associate with heparin and is not a mitogen for either BALB/c 3T3 cells or human umbilical vein ECs.15 This implies two possibilities: (1) either dimerization of FGF-1 that accompanies temperature-induced FGF-1 secretion and is connected with active transmembrane transportation of the protein15 50 does not occur with oxLDL or (2) monomerization of FGF-1 may be a secondary, postsecretory event in which oxLDL acts as a reductant. There may be other interactions between the oxLDL and the released growth factor, as suggested by the slightly different size of the oxLDL-released FGF-1.

Furthermore, inhibitors of RNA and protein synthesis, actinomycin D and cycloheximide, respectively, did not affect the release of FGF-1 in response to oxLDL. However, with temperature stress, these inhibitors substantially reduced the exit of FGF-1 into culture medium.15 These findings led to a conclusion that temperature either upregulates the expression of FGF-1 itself or influences posttranslational regulatory factors involved in mediating the FGF-1 secretion.14 15 50 Our data suggest that FGF-1 released in response to oxLDL comes from the presynthesized cytoplasmic pool and does not require de novo synthesis of either FGF-1 itself or other proteins necessary for its release from the cell.

Finally, brefeldin A, a pharmacological agent known to impair the secretory function of the ER Golgi apparatus,62 potentiated rather than repressed the secretion of FGF-1 in response to elevated temperature50 but had no significant effect on FGF-1 release in response to oxLDL, confirming that a pathway other than the classic one must be involved in both cases.

The secretion of FGF-121-154 in response to elevated temperature in a latent form is proposed to be a mechanism specific for FGF-1, since heat shock does not promote secretion of the other prototype member of FGF family, FGF-2.25 Further investigations are needed to examine the specificity of oxLDL-induced FGF-1 release. However, the data obtained led us to the conclusion that some more general process, such as sublethal and probably transient and reversible cell injury, may be involved. Several lines of evidence support this hypothesis. (1) Light and electron microscopic analysis of cell morphology, trypan blue exclusion tests, LDH measurements, MTT-based viability assay, and replating studies show that FGF-1 release is not a reflection of lethality caused by toxicity of oxLDL. (2) Our calculations of LDH activity and the amount of FGF-1 released showed that >90% of the FGF-1 that had accumulated in the conditioned medium appeared to be released by viable but nonlethally damaged cells, probably through transient changes in cell membrane permeability. Interestingly, these alterations in membrane permeability favor the selective release of FGF-1 relative to LDH, because the ratio of LDH to FGF-1 in releasate was almost twice as low as that in the cell lysate. (3) FGF-1 release was maximal during the first 4 hours of incubation with oxLDL and did not increase over the next 12 hours. These results suggest a temporary and reversible character of cell membrane changes imposed by oxLDL. However, this speculation is based on indirect evidence, and the possibility that other mechanisms may underlie this observation cannot be excluded. (4) The amount of FGF-1 released correlated with an increase in cell membrane permeability to a low-molecular-weight marker, PI.

Considerable evidence supports the concept that sublethal changes in cell membrane properties may be a physiologically relevant mode of the release of growth factors. Most of these data refer to the release of FGF-2 that can be induced by chemically or physically injurious agents that model wounding of cells in vivo. Treatment of bovine aortic ECs with endotoxin releases FGF-2.24 De novo synthesis and release of FGF-2 have been reported to occur as part of the cellular responses to irradiation.19 Scraping of ECs leads to the release of 40% to 80% of cell-associated FGF-2 and induces a significant (4- to 10-fold) increase in steady-state FGF-2 mRNA levels.17 18 20 Interestingly, these survivable disruptions of the plasma membrane favor the selective release of the growth factor relative to other cytosolic molecules that are freely diffusible from damaged cells, such as LDH.20 This is consistent with our findings for FGF-1. Transient alterations in sarcolemmal permeability of paced adult rat ventricular myocytes resulted in the release of cytosolic FGF-2 and probably FGF-1.21 22 Activation of the cultured ECs by mildly oxidized LDL increased the level of expression of a 14.5-kD endogenous lectin, galectin-1, which also lacks the signal sequence for secretion.64 Benzaquen et al16 showed that sublytic concentrations of the complement C5b-9 complex release a mitogenic FGF-2 from human umbilical vein and bovine aortic ECs into culture medium without evidence of severe cell injury or cell death, implying a transient and very short-lived character of membrane lesions.

We have demonstrated that oxLDL is able to induce the release of FGF-1 from NIH 3T3 fibroblasts and rabbit SMCs expressing elevated levels of this growth factor. We propose that in the atherosclerotic lesion, oxLDL may lead to the local release of cytosolic FGF-1 in the mitogenically active form from lesional macrophages, SMCs, or other FGF-1–producing cells. This release is apparently mediated by a sublethal and probably reversible injury to the cell membrane that is consistent with the observation that no massive cell death occurs in early lesions. We propose that in addition to cytokine release caused by stimulation of protein synthesis and secretion,30 oxLDL-induced FGF-1 release may be another pathogenic mechanism stimulating endothelial and SMC proliferation in such disorders as atherosclerosis.


*    Selected Abbreviations and Acronyms
 
EC = endothelial cell
ECL = enhanced chemiluminescence
ER = endoplasmic reticulum
FGF = fibroblast growth factor
FGF-1 = acidic fibroblast growth factor
FGF-2 = basic fibroblast growth factor
LDH = lactate dehydrogenase
oxLDL = oxidized LDL
PI = propidium iodide
SFM = serum-free growth medium
SMC = smooth muscle cell
TBS-Tw = Tris-buffered saline with Tween 20


*    Acknowledgments
 
This study was supported by grants HL-54246, HL-29582, HL-47852, HL-30568, HL-37510, and HL-39727 from the National Institutes of Health. We are indebted to Dr X. Zhan (Department of Experimental Pathology, Holland Laboratories, American Red Cross, Rockville, Md) for providing NIH 3T3 cell pMEXneo:FGF-1 transfectants and to Dr John A. Thompson (Department of Surgery, University of Alabama at Birmingham) for supplying us with a preparation of human recombinant FGF-1 and rabbit anti-human FGF-1 antibody. We express our special gratitude to Yamei Gao for performing electron microscopy studies. We also thank Pamela Kristie Thompson for active technical assistance and Diana Norman for her help with photography.

Received February 24, 1996; accepted July 11, 1996.


*    References
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