Cytosolic 85-kDa Phospholipase A2-mediated Release of Arachidonic Acid Is Critical for Proliferation of Vascular Smooth Muscle Cells*

Recent evidence suggests that arachidonic acid (AA) may be involved in regulating cellular proliferation. The predominant mechanism of AA release from cellular phospholipids is via phospholipase A2 (PLA2) hydrolysis. The purpose of this study was to examine the roles of the distinct 14-kDa and 85-kDa PLA2 enzymes in human coronary artery vascular smooth muscle cell (hCAVSMC) proliferation. Cultured hCAVSMCs proliferate in the presence of growth medium with a typical doubling time of 30–40 h, grow at a slower proliferative rate upon reaching confluency (day 8), and eventually undergo contact inhibition of growth (day 10). Neither Type II 14-kDa PLA2activity nor mass changed over a 10-day culture period. In contrast, 85-kDa PLA2 protein activity and mRNA decreased as time in culture progressed. This reduction in 85-kDa PLA2correlated with reductions in DNA synthesis and suggested a possible association between 85-kDa PLA2 and proliferation. To directly evaluate the role of the 85-kDa PLA2 in proliferation we examined the effects of an 85-kDa PLA2inhibitor (AACOCF3) and 85-kDa PLA2 antisense oligonucleotides on proliferation. Both reagents dose dependently inhibited proliferation, whereas a 14-kDa PLA2 inhibitor (SB203347), a calcium-independent PLA2 inhibitor (HELSS), an 85-kDa sense oligonucleotide, and a nonrelevant scrambled control oligonucleotide had no effect. The mechanism by which 85-kDa PLA2 influences cellular proliferation remains unclear. Inhibition of 85-kDa PLA2 activity produced neither phase-specific cell cycle arrest nor apoptosis (fluorescence-activated cell sorter analysis). Addition of AA (20 μm) attenuated the effects of both AACOCF3 and 85-kDa antisense oligonucleotides implicating AA as a key mediator in cellular proliferation. However, although prostaglandin E2(PGE2) was present in the culture medium, it peaked early (day 3) in culture, and indomethacin had no effect on cellular proliferation indicating that hCAVSMC proliferation was not mediated through PGE2. These data provide the first direct evidence that PLA2 is involved in control of VSMC proliferation and indicate that 85-kDa PLA2-mediated liberation of AA is critical for cellular proliferation.

Proliferation of vascular smooth muscle cells (VSMCs) is implicated in the pathogenesis of hypertension, primary atherosclerosis, and restenosis following interventional revascularization procedures such as balloon angioplasty, arterial stenting, and by-pass surgery (33)(34)(35). Evidence suggestive of a role for PLA 2 in VSMC proliferation includes: 1) that exogenous administration of AA increases expression of the early response genes c-myc (36), c-fos (36), and c-jun (37), as well as activity of mitogen-activated protein kinase (38); and 2) that these effects as well as serum-and growth factor-induced proliferation are inhibited by nonselective PLA 2 inhibitors such as mepacrine (1,37,39).
VSMCs exhibit both 14-kDa and high molecular mass cytosolic PLA 2 activities (24,40). Recent descriptions of hydrogen peroxide-stimulated DNA synthesis and expression of proliferation-associated early response genes (36 -38, 41), and hydrogen peroxide- (42) and angiotensin II-mediated (43) phosphorylation of the 85-kDa PLA 2 infer that this enzyme may participate in AA release during cellular proliferation. Other data (44,45) demonstrate that vascular smooth muscle cells possess high affinity Type I PLA 2 -specific binding sites and that 14-kDa phospholipase A 2 is present in atherosclerotic plaques (46,47), suggesting that the 14-kDa PLA 2 serves a function in processes related to vascular disease. But, whether the 14-kDa PLA 2 plays a role in cellular proliferation is not known. The purpose of this study was to directly examine the roles of the distinct 14-and 85-kDa PLA 2 enzymes in VSMC proliferation. Herein is presented the first direct evidence that PLA 2 activity is associated with VSMC proliferation. Neither the 14-kDa PLA 2 nor prostaglandin E 2 (PGE 2 ) appear to mediate VSMC proliferation. In contrast, the 85-kDa enzyme appears to serve a selective role in the initial production of arachidonic acid, which, through direct action or via action of metabolites other than PGE 2 , is critical for VSMC proliferation.

Cell Culture
Human coronary artery vascular smooth muscle cells (hCAVSMCs, Clonetics, San Diego, CA) were plated at 3 ϫ 10 3 cells/cm 2 and grown in monolayer in the optimized culture medium recommended by the manufacturer (Clonetics SmGM2: modified MCDB131 containing fetal bovine serum (5%), insulin (5 mg/ml), fibroblast growth factor (2 ng/ml), epidermal growth factor (0.5 mg/ml), gentamycin (50 mg/ml), and amphotericin ␤ (50 ng/ml)) at 37°C in an atmosphere of 5% CO 2 , 95% air and 95% humidity. Cells were passaged when 70 -80% confluent via a 4-min treatment (37°C) with trypsin-EDTA (0.01:0.02%) in calciummagnesium-free phosphate-buffered saline containing (in mM): NaCl (137); Na 2 HPO 4 (8.1); KCl (2.7); and KH 2 PO 4 (1.5), pH 7.4. Cells were reseeded into 24-well plates, 150-or 600-cm 2 culture flasks at 3 ϫ 10 3 cells/cm 2 . The medium was changed every third day, and these asynchronous cultures of randomly dividing cells were allowed to grow for various times as indicated. Our goal was to take advantage of the fact that these cells routinely demonstrate reduced DNA synthetic rate upon reaching confluency and contact inhibition within several days of reaching confluency. All experiments were done using cells from passages 5-7 and were done using two different hCAVSMC lines to rule out donor-specific results. Microscopic examination of cellular morphology was done at 40ϫ magnification with an inverted phase-contrast microscope (Telaval 31, Carl Zeiss, Inc., NY) equipped with a camera port for photographic documentation.
Normal Growth of hCAVSMCs-Normal growth curves were determined by allowing the cells to grow for 14 days, which, based on prior experience in our laboratory, was expected to span the entire proliferative phase of these cell lines under the present culture conditions. At various times, the cultures were washed with calcium-magnesium free phosphate-buffered saline, harvested with trypsin-EDTA, diluted with culture medium, and counted by hemacytometer. In parallel wells, relative rates of DNA synthesis were assessed via [ 3 H]thymidine incorporation (4-h pulse) into trichloroacetic acid precipitable material as described previously (48). [ 3 H]Thymidine incorporation was adjusted for cell density at the time the pulse was given.

Smooth Muscle Subcellular Fractionation
Preconfluent (day 3; i.e. actively proliferating), near or at confluent (ϳday 8), and postconfluent (days 10 -14) cells were harvested by trypsinization and centrifugation. The smooth muscle cell pellet (2.6 -5.7 ϫ 10 7 cells) was resuspended to 1 ϫ 10 8 cells/ml of homogenization buffer containing 0.34 M sucrose, 10 mM HEPES, pH 7.4, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 200 M leupeptin, 20 g/ml soybean trypsin inhibitor, and 20 g/ml aprotinin at 4°C. Inclusion of EGTA localized the 85-kDa PLA 2 predominantly in the cytosolic fraction (50,51). The cell suspension was disrupted by nitrogen cavitation (450 p.s.i. for 15 min at 4°C), and the homogenate was centrifuged at 400 ϫ g for 10 min at 4°C to remove unbroken cells and debris. The resulting supernatant fraction was centrifuged at 100,000 ϫ g for 60 min at 4°C to obtain a supernatant (cytosolic) and particulate (microsomal) fraction. The microsomal pellet was resuspended in homogenization buffer, and both fractions were flash frozen with liquid N 2 , and stored at Ϫ70°C until analysis.

Phospholipase A 2 Enzyme Assay
Phospholipase A 2 activity of hCAVSMC subcellular fractions (50 -100 g of protein/assay) was measured by the acylhydrolysis of [ 3 H]AA-Escherichia coli (NEN Life Science Products) or [1-14 C]palmitoyl-2arachidonyl phosphatidylcholine ([ 14 C]AA-PC, Amersham Corp.) as described previously (14). The assay was initiated by addition of substrate, and assays were incubated at 37°C for a time predetermined to be on the linear portion of a time versus hydrolysis plot. Specific activity (picomoles of free fatty acid hydrolyzed/minute/milligram) was determined from percent hydrolysis values.

Flow Cytometry
Apoptosis was measured by TUNEL (56,57), using the ApopTag kit from Oncor (Gaithersburg, MD). In brief, the enzyme terminal deoxynucleotidyltransferase extends the DNA fragments with digoxigenin-containing nucleotides, which are then detected with a anti-digoxigenin antibody carrying fluorescein to allow detection by fluorescence (494-nm excitation, 523-nm emission). Propidium iodide was used as a counterstain to measure total DNA content and to determine distribution of cells in G 0 /G 1 , S, and G 2 /M phases of the cell cycle. Flow cytometric analysis was performed on a Becton Dickinson FACScan instrument using CellQuest software.

Eicosanoid Measurement
PGE 2 , PGF 2␣ , leukotriene B4, and leukotriene C4 levels in cell-free medium were directly measured using enzyme immunoassay (Cayman Chemical Co.). Sample or standard dilutions were made with SmGM2 and analyzed in triplicate.

Protein Determination
All protein concentrations were determined by Bradford protein analysis kits (Bio-Rad).

Data Analysis
Data are expressed as mean Ϯ S.D. or standard error of the mean (S.E.) as indicated. Each data point represents n ϭ 2-3 unless otherwise stated. Individual statistical comparisons of paired data were evaluated by Student's t test with p Ͻ 0.05 representing significance.

Normal Growth of Human Coronary Artery Vascular Smooth Muscle Cells
Cells were plated at 3 ϫ 10 3 cells/cm 2 (6000 cells/well) on day 0 and maintained in normal growth medium as described under "Experimental Procedures." On days 3 (sparse), 8 (near confluent), 11 (confluent), and 14 (postconfluent) cells were harvested for determination of cell number. As shown in Fig. 1, absolute cell number increased as a function of time. Analysis of the data revealed that cell number increased 2.2-fold, 2.5fold, and 1.2-fold between days 0 and 3, 8 and 11, and 11 and 14, respectively, reflecting the expected reduction in cellular proliferative rate in postconfluent cells. Parallel wells were pulsed with [ 3 H]thymidine to measure relative rates of DNA synthesis. Incorporation of the radiolabeled thymidine was normalized to the number of cells present during the radioactive pulse. [ 3 H]thymidine incorporation was highest in sparsely seeded, robustly proliferating cells (day 3) (Fig. 1) and decreased with time in culture, with little or no [ 3 H]thymidine incorporation evident in postconfluent (day 14) cells. Thus, as expected, DNA synthesis decreased prior to the reduction in cellular proliferative rate, and ultimately proliferation slowed in postconfluent cells. These observations are consistent with postconfluent contact-inhibited reduction in cellular growth. Subsequent studies were done using hCAVSMCs cultured to these same timepoints unless otherwise stated.

Eicosanoid Measurements
The hCAVSMCs used in these studies produced PGE 2 and PGF 2␣ as determined by its presence in the culture medium (ELISA). PGE 2 levels increased from day 0 to day 3, but did not change over days 3-14 of the culture period: pg/ml/cell, 2.08 Ϯ 0.25 (n ϭ 2, day3); 2.18 Ϯ 1.01 (n ϭ 2, day 8); and 1.76 (n ϭ 1, day 10). Indomethacin (1 M) produced 87.3 Ϯ 2.04 percent reduction in PGE 2 synthesis (mean Ϯ S.E., n ϭ 3 in duplicate). The levels of PGF 2␣ were comparable to the levels of PGE 2 detected in the same samples. Given that PGE 2 synthesis was inhibited by indomethacin, one can assume that indomethacin had equivalent effects on PGF 2␣ synthesis as has been previously described in a number of cell systems (58). These cells did not produce detectable levels of leukotriene B4 or leukotriene C4 (data not shown).

Analysis of 85-kDa PLA 2 and 14-kDa PLA 2 as a Function of Culture Period
Measurement of Enzyme Activity-Human CAVSMCs were harvested and homogenized, and subcellular fractions were made from preconfluent (day 3), near or at confluent (days 7-8), and postconfluent (days 14 -15) cultures as described under "Experimental Procedures." Cell fractions were assayed for acylhydrolytic activity of 85-kDa PLA 2 activity via using [1-14 C]palmitoyl-2-arachidonyl phosphatidylcholine as described under "Experimental Procedures." As shown in Fig. 2, cytosolic 85-kDa PLA 2 activity was highest on day 3 commensurate with the highest proliferative rate. Activity steadily decreased from day 3 to day 14 coincident with the reduction in the relative rate of DNA synthesis over this culture period. The 85-kDa PLA 2 activity in the smooth muscle microsomal fraction was very low and remained constant over the length of the culture, indicating that the reduced cytosolic 85-kDa PLA 2 activity was not due to migration of protein to microsomes. 14-kDa PLA 2 activity measured by acylhydrolysis of

85-kDa PLA 2 Is Critical for Cellular Proliferation
sequently subjected to SDS-polyacrylamide gel electrophoresis and probed with anti-85 kDa PLA 2 rabbit serum. As shown in Fig. 3 (panels A and B), the hCAVSMC cytosolic fractions from two separate studies possessed a high molecular weight protein that was recognized by rabbit anti-85 kDa PLA 2 serum and co-migrated characteristically with the rh 85-kDa PLA 2 protein at 110 kDa. The immunoreactive bands decreased in intensity from day 3 through day 14 indicating a decrease in protein over the culture period. Microsomal fractions contained immunoreactive 85-kDa PLA 2 bands, but band intensity did not change as a function of culture period. In a separate study (Fig. 3C) the 85-kDa PLA 2 protein was evaluated in cells cultured between 3 h and 10 days. Western analysis of cytosolic fractions demonstrated that the 85-kDa PLA 2 protein was present in cells that had been incubated for only 3 h, that the 85-kDa PLA 2 protein level increased between 3 h and day 1, and was further increased on day 3. Levels subsequently decreased by day 10 in agreement with the data shown in Fig. 3, A and B. Northern analysis of hCAVSMCs in parallel culture dishes is shown in Fig. 4. RNA from two independent studies was fractionated on the same gel. Hybridization indicates that the 85-kDa PLA 2 message is present at day 3 and that it decreases with increased time in culture. This supports that the reduction in 85-kDa PLA 2 protein level from day 3 to day 14 was due, at least in part, to a decrease in 85-kDa PLA 2 message.
Assessment of 14-kDa PLA 2 Mass by ELISA-Analysis of the subcellular fractions by ELISA indicated the presence of low levels of type II 14-kDa PLA 2 immunoreactive protein in the cytosol and approximately 10-fold greater levels in the particulate fraction, but no significant change in the 14-kDa PLA 2 protein was observed over the culture period (Table I).

Effect of PLA 2 Modulation on hCAVSMC Proliferation
The Effect of PLA 2 Inhibitors-To further examine the nature of the association of 85-kDa PLA 2 with cellular proliferation and to examine whether 85-kDa PLA 2 may have an active role in regulation of VSMC proliferation, we evaluated the effects of 85-kDa PLA 2 , 14-kDa PLA 2 , and iPLA 2 inhibitors on cellular proliferation. AACOCF 3 , a trimethyl ketone analogue of arachidonic acid recently shown to inhibit human recombinant 85-kDa PLA 2 and cell-associated 85-kDa PLA 2 (59, 60) was added to day 3 cultures at different concentrations (0 -10 M) and further incubated for 3 days. Relative to the vehicle (0.03% ethanol) control cells, this compound dose dependently

FIG. 3. Western analysis of microsomal and cytosolic fractions of human VSMCs cultured 0 -14 days is shown in panels A and B.
Smooth muscle subcellular fractions were subjected to SDS-polyacrylamide gel electrophoresis (10%), transferred to nitrocellulose, and blotted with rabbit antisera NS1 made against the 85-kDa PLA 2 ; duplicate independent experiments run on different hCAVSMC cell lines. S indicates the rh U937 85-kDa PLA 2 used as a standard (0.86 g of cytosolic protein); numbers indicate days in culture (3,7,8,14,15). Lanes were equally loaded with 50 g of total protein per lane. Smooth muscle cytosolic fractions (20 g/lane) blotted with 85-kDa PLA 2 antisera is shown in panel C. S, standard; lanes indicate cytosol from cells obtained at 3 h (3 h), 1, 3, and 10 days (1d, 3d, and 10d) after seeding.

FIG. 4. Northern analysis of 85-kDa PLA 2 mRNA in cells cultured over time.
Random-primed radiolabeled full-length 85-kDa PLA 2 cDNA probe (specific activity Ͼ 1 ϫ 10 9 cpm/g) was hybridized to electrophoretically separated total RNA (10 g/lane) and washed under high stringency conditions. Lanes 1 and 4, day 3 cultures; lanes 2 and 5, day 8 cultures; lanes 3 and 6, day 14 cultures. A, single bands detected with cDNA probes for 85-kDa PLA 2 (3.4 kb) and GAPDH (1.4 kb, to normalize for loading variation); B, ratio of the intensity of the 85-kDa PLA 2 hybridization signal to the intensity of the GAPDH hybridization signal was calculated for each lane from data obtained by phosphorimager analysis. This data was averaged from the two independent experiments and expressed graphically.

85-kDa PLA 2 Is Critical for Cellular Proliferation
inhibited hCAVSMC proliferation (Fig. 5). Recall that these cells were seeded at a density of 6000 cells/well on day 0 and that by day 3, the cell number had increased 2.2-fold to 13,209 Ϯ 200 cells/well (Fig. 1). Absolute cell number following the 3-day incubation period with 10 M AACOCF 3 was 12,472 Ϯ 788.2 (mean Ϯ S.D.) indicating that this concentration of AACOCF 3 completely inhibited cellular proliferation, and that the apparent IC 50 of AACOCF 3 in our system is Х1 M (Fig. 5). In contrast, neither the selective 14-kDa PLA 2 inhibitor, SB203347 (0 -10 M) (59, 61), nor the cyclooxygenase inhibitor, indomethacin (1 M), had significant effects on hCAVSMC proliferation. The long doubling time of these cells required that long incubation times be used to ensure a sufficient window over which to observe changes in proliferation. A possible concern is that the lack of an effect of the SB compound on hCAVSMC proliferation may be due in part to a possible breakdown of the drug over the 3-day incubation period used in these studies. However, 1 and 2 days of exposure to SB203347 (10 M) produced no reduction in cell number (97.9 and 93.3% of control, respectively). Thus, the lack of an effect of SB203347 reported in Fig. 5 is not likely due to compound instability. Morphologic examination (Fig. 6) demonstrated that the various drug treatments produced no obvious changes in cell structure, and trypan blue exclusion methodology determined that cell viability in AACOCF 3 -and SB203347-treated cells (10 M) was Ͼ95% and was equivalent to vehicle (ethanol) control cells. Thus, AACOCF 3 -mediated reduction of cellular growth was not due to cellular toxicity. Higher Effects of 85-kDa PLA 2 Antisense and Exogenous Arachidonic Acid on hCAVSMC Proliferation-Previous work in this laboratory utilized 85-kDa PLA 2 initiation site-directed antisense oligonucleotides in human monocytes (31,59) and human synovial fibroblasts (49,62) to assess the role of this enzyme in prostanoid and leukotriene production. To more specifically examine the nature of the regulation of cellular proliferation by 85-kDa PLA 2 , hCAVSMCs were seeded (day 0), allowed to adhere overnight and then incubated (18 -24 h, 37°C) in growth factor-and antibiotic-free medium in the presence of increasing concentrations (0.01-1.0 M) of antisense to 85-kDa PLA 2 (SB7111), complementary sense oligonucleotide (SB9030, 0.01-1.0 M), a scrambled nonrelevant oligonucleotide control (SB7222, 1 M), or Lipofectin vehicle as described under "Experimental Procedures." Following this preincubation, growth medium (SmGM2) was added. For comparison, some cells received AACOCF 3 (3 M). To evaluate the role of AA in 85-kDa PLA 2 -mediated effects on proliferation, parallel wells were incubated with AA (20 M) alone, AA together with antisense oligonucleotides or with AACOCF 3 . Following a 3-day incubation period, cell viability and cell number were determined. As shown in Table II, Lipofectin alone had no effect on cell number. SB7111 concentration dependently inhibited growth medium-induced proliferation with both 0.1 and 1.0 M reaching statistical significance at p Ͻ 0.05. Recall that these cells were seeded at a density of 5 ϫ 10 4 cells/well prior to transfection and the 3-day exposure to growth medium to stimulate proliferation. Absolute cell number following this 3-day stimulation in the wells treated with 1.0 M SB7111 was 4.47 Ϯ 0.9 ϫ 10 4 (mean Ϯ S.D.) indicating that this concentration of the antisense oligonucleotide completely inhibited cellular proliferation. The sense control (SB9030) and scrambled nonrelevant

85-kDa PLA 2 Is Critical for Cellular Proliferation
oligonucleotides had no effect (Table II). Addition of 20 M AA itself directly and significantly stimulated cellular proliferation (Table II). Exogenous AA attenuated the anti-proliferative effect of SB7111 as evidenced by only 10 and 20% reductions in cell number in the presence of both AA and SB7111 (0.1 and 1.0 M, respectively) versus 37 and 55% reductions in the presence of SB7111 alone. In agreement with other data presented herein (Fig. 5) AACOCF 3 (3.0 M) inhibited growth factorinduced proliferation by 47%. This was significantly attenuated by addition of AA as indicated by only 9% inhibition of proliferation during co-incubation of 20 M AA and AACOCF 3 . Trypan blue exclusion and close visual inspection of cell morphology indicated that the observed decreases in cell number following exposure to SB7111 or AACOCF 3 were not due to cytotoxicity.

Flow Cytometry
Fluorescence-activated cell sorter (FACS) analysis was performed to evaluate the effect of AACOCF 3 on the cell cycle. Cells incubated with 10 M AACOCF 3 (the highest concentration used in the proliferation studies described above) showed no evidence of morphological changes and no overt cytotoxity but had reduced cell number as expected (42% of non-AA-COCF 3 -treated controls). Cells were collected and stained with propidium iodide and TUNEL after 1, 2, and 3 days of exposure to the drug. Table III provides data from one representative of two experiments that shows that 10 M AACOCF 3 caused no remarkable change in the distribution of cells in G 0 /G 1 , S, and G 2 /M phases over 3 days. The left panel of Fig. 7 shows the counts versus propidium iodide intensity and graphically de-picts the numerical data for the cell cycle stages given in Table  III; again, no difference was observed in the distribution of the cells in AACOCF 3 -treated cells. The right panel of Fig. 7 shows that 3 days of AACOCF 3 exposure produced no DNA fragmentation (e.g. no apoptosis) as evidenced by no movement of signal into quadrant R3. A shift of cells into the R3 quadrant is typically observed in our laboratory with anti-FAS antibodyinduced apoptosis in Jurkat cells and is routinely reported for other apoptotic cells (57). DISCUSSION Although previous studies demonstrated that nonselective PLA 2 inhibitors reduce VSMC proliferation (36 -39, 42, 43), the present study represents the first direct examination of the two distinct PLA 2 enzymes in VSMC proliferation. Moreover, it is the first study that cleary demonstrates in any cell type that the 85-kDa PLA 2 and not the 14-kDa PLA 2 is critical for cellular proliferation. Herein it is confirmed that vascular smooth muscle cells possess both the 14-kDa PLA 2 and 85-kDa PLA 2 enzymes. By taking advantage of contact inhibition of cellular growth despite continual presence of growth medium, it was first demonstrated that 85-kDa PLA 2 activity, protein level and mRNA expression (but not 14-kDa PLA 2 activity or mass) correlated with DNA synthesis. 85-kDa PLA 2 levels were highest early in culture and rose to a maximum level and activity at day 3, which corresponds with the peak in [3H]thymidine uptake, indicating an association between 85-kDa PLA 2 and cellular proliferation and suggesting an important role of this protein in robustly proliferating hCAVSMCs.
To further and more directly evaluate the role of 85-kDa PLA 2 in hCAVSMC proliferation, PLA 2 inhibitors and antisense oligonucleotides were used. In both AACOCF 3 -and SB7111-treated cells, replication assessed by cell counts was concentration dependently reduced relative to vehicle control. Indeed, at the highest concentration of SB7111 cell numbers at day 3 did not vary from the initial seeding density suggesting that SB7111 produced complete cessation of cell cycle activity and thereby completely inhibited cellular proliferation. Again, neither SB203347, HELSS, nor SB9030 affected proliferation. Taken together, these data further support an important role of the 85-kDa PLA 2 but not the 14-kDa PLA 2 nor iPLA 2 in hCAVSMC proliferation.
FACS scan analysis suggests that the reduction in cell number following inhibition of 85-kDa PLA 2 results from generalized attenuation of cellular proliferative rate rather than phase-specific cell cycle arrest. Furthermore, the AACOCF 3treated cells showed no indication of entering programmed cell death. Similiar results were observed in HL-60 cells exposed to AACOCF 3 (63). The present data may offer an explanation for a previous observation (64); increased PLA 2 activity is readily observed following stimulation of proliferating cultures of murine embryo palate mesenchymal cells with the calcium ionophore A23187, but confluent cultures showed no such response. Based on our findings, we hypothesize that the confluent mu-   (10 M) produces no phase-specific arrest of the cell cycle Cells were trypsinized, seeded into 150-cm 2 culture flasks, allowed to adhere for 4 h in normal growth medium (SmGM2, control) before addition of AACOCF 3 (10 M), and then incubated in the presence of a drug for 1, 2, or 3 days. Effect of AACOCF 3 on hCAVSMC cell cycle was examined by FACS scan analysis of propidium iodide-labeled cells. The data are from one representative of two experiments and are expressed as percent of total cells in the particular phase of the cell cycle. Reduction in cell number (cell counts, hemacytometer) obtained from parallel cultures verified AACOCF 3  The mechanism by which 85-kDa PLA 2 influences cellular proliferation remains to be determined. PGE 2 was produced by hCAVSMCs between day 0 and day 3, but PGE 2 levels did not change further with changes in proliferation. This is consistent with the coordinate early rise in 85-kDa PLA 2 protein during the same time frame and suggests a possible role of PGE 2 in the initial phase of proliferation. However, indomethacin had no effect on cellular proliferation under the conditions of our experiments, indicating that the 85-kDa PLA 2 dependent effect on VSMC proliferation is not mediated through PGE 2 . This is in agreement with previous reports that cyclooxygenase inhibition by indomethacin had no effect on VSMC proliferation (1, 39), AA-, or hydrogen peroxide-induced c-jun expression in VSMCs (37) or AA activation of mitogen-activated protein kinase (38). When cells were treated with exogenous AA, the reduction in cell number produced by either AACOCF 3 or 85-kDa PLA 2 antisense oligonucleotides (SB7111) was prevented or significantly attenuated, suggesting that the 85-kDa PLA 2 may influence cellular proliferation largely through its generation of free AA. Interestingly, 20 M AA by itself stimulated hCAVSMC proliferation, increasing the cell number 9% above untreated controls. The exact role of AA is not known, but as discussed above, conversion to PGE 2 , LTB 4 , and LTC 4 does not seem to be obligatory for proliferation to proceed. This raises the possibility that AA is itself an intracellular signal and/or that it is converted to an unidentified eicosanoid product important in regulation of cellular proliferation. It is possible that the present demonstration of a direct proliferative effect of exogenous administration of AA to hCAVSMCs and attenuation of AACOCF 3 -and 85-kDa PLA 2 antisense-mediated reductions in proliferation by AA may be related to the effect of AA to stimulate c-fos, c-jun, and/or mitogen-activated protein kinase (36 -39, 42, 43).
The mechanism by which 85-kDa PLA 2 influences cellular proliferation may be related to previous demonstrations that inhibition of the lipoxygenase-cytochrome P 450 system reduces VSMC proliferation or proliferation-associated events (1,(37)(38)(39) and/or that 85-kDa PLA 2 is activated by oncogenic ras (65,66). Alternatively, or additionally, recent reports indicate that the release of AA by PLA 2 is accompanied by the formation of biologically active lysolipids, and that lysophosphatidic acid, lysophosphatidylinositol, and lysophosphatidylcholine stimulate mitogen-activated protein kinase activity and proliferation in a variety of cell types (67)(68)(69)(70)(71). Furthermore, these lysolipids serve as precursors to a new class of biologically active lipid derivatives, the glycerophosphoinositides, which show evidence of being accumulated specifically in ras-transformed cells. Given that the present data indicate that it is the 85-kDa PLA 2 that selectively regulates the initial release of AA to influence cellular proliferation, it would be interesting to examine the effects of AACOCF 3 or 85-kDa antisense on accumulation of these lipids in ras-transformed cells.
Rao et al. (37) report that nonselective PLA 2 inhibition by mepacrine either had no effect on c-jun expression induced by platelet-derived growth factor, partially attenuated epidermal growth factor-stimulated c-jun expression, or significantly inhibited hydrogen peroxide-induced c-jun expression in VSMCs suggesting a potential growth factor-specific nature to the role of PLA 2 in VSMC proliferation. To avoid complications associated with specific growth factors, the present studies were designed to take advantage of contact inhibition of cellular growth despite continual presence of growth medium to define whether the 14-and 85-kDa PLA 2 enzymes are involved in regulation of cellular proliferation. Thus, the present study routinely involved stimulation of hCAVSMC proliferation by a defined optimized medium supplied by the manufacturer that contained fetal bovine serum (5%), insulin (5 mg/ml), fibroblast growth factor (2 ng/ml), and epidermal growth factor (0.5 mg/ ml). Accordingly, we cannot comment on whether the 85-kDa PLA 2 serves a particular role in the proliferative response to these specific growth factors. Examination of this issue provides the basis for future additional studies.
The present finding that 85-kDa PLA 2 is apparently required for cellular proliferation may be relevant to pathologies associated with VSMC hyperplasia. In the normal noninjured vessel, smooth muscle cells have a low mitotic rate (72). The VSMC hyperplasia evident in atherosclerotic and restenotic vessels is believed to be part of a sequelae of responses to vascular injury by numerous agents including viruses, caustic by-products of cigarettes, hypercholesterolemia, and physical injury. PLA 2 activation has been associated with cellular injury in kidney, heart and liver (71). It remains to be determined if cellular injury specifically alters 85-kDa PLA 2 expression and activity and if the 85-kDa PLA 2 is up-regulated in atherosclerotic or restenotic vessels. Nevertheless, the present data support the hypothesis that selective inhibitors of 85-kDa PLA 2 may prove effective therapeutics for treatment of vascular disease associated with aberrant VSMC proliferation.
Acknowledgments-We thank Brian Bolognese and Michael Hansbury for performing the PGE 2 and FACS analysis studies, respectively. . Cells were fixed with 2% formalin and stained with propidium iodide to measure total DNA content, and the TUNEL method was used to measure DNA fragmentation as described under "Experimental Procedures." The left panel shows the relative number of cells versus propidium iodide fluorescence (FL2-A); the typical pattern of cell cycle distribution (G 0 /G 1 , S, and G 2 /M) was not significantly different between control and AACOCF 3 -treated cells (refer to Table III for numerical percentages of these distributions). The right panel shows the results of individual cells for propidium iodide fluorescence versus the TUNEL fluorescence (FL1-H). The boxes indicate cells that are apoptotic (R3) or apoptotic/dead (R4); 3 days of exposure to 10 M AACOCF 3 produced no increase in the number of apoptotic or dead cells.