Prostaglandin D2 synthase inhibits the exaggerated growth phenotype of spontaneously hypertensive rat vascular smooth muscle cells.

Lipocalin-type prostaglandin D2 synthase (L-PGDS) has recently been linked to a variety of pathophysiological cardiovascular conditions including hypertension and diabetes. In this study, we report on the 50% increase in L-PGDS protein expression observed in vascular smooth muscle cells (VSMC) isolated from spontaneously hypertensive rats (SHR). L-PGDS expression also increased 50% upon the differentiation of normotensive control cells (WKY, from Wistar-Kyoto rats). In addition, we demonstrate differential effects of L-PGDS treatment on cell proliferation and apoptosis in VSMCs isolated from SHR versus WKY controls. L-PGDS (50 microg/ml) was able to significantly inhibit VSMC proliferation and DNA synthesis and induce the apoptotic genes bax, bcl-x, and ei24 in SHR but had no effect on WKY cells. Hyperglycemic conditions also had opposite effects, in which increased glucose concentrations (20 mm) resulted in decreased L-PGDS expression in control cells but actually stimulated L-PGDS expression in SHR. Furthermore, we examined the effect of L-PGDS incubation on insulin-stimulated Akt, glycogen synthase kinase-3beta (GSK-3beta), and ERK phosphorylation. Unexpectedly, we found that when WKY cells were pretreated with L-PGDS, insulin could actually induce apoptosis and failed to stimulate Akt/GSK-3beta phosphorylation. Insulin-stimulated ERK phosphorylation was unaffected by L-PGDS pretreatment in both cell lines. We propose that L-PGDS is involved in the balance of VSMC proliferation and apoptosis and in the increased expression observed in the hypertensive state is an attempt to maintain a proper equilibrium between the two processes via the induction of apoptosis and inhibition of cell proliferation.

The pathogenesis of cardiovascular disease involves the abnormal accumulation of vascular smooth muscle cells (VSMCs) 1 within the intima of blood vessels due to a shift in the equilibrium between cell proliferation, growth, and apoptosis (1,2). Recently, we demonstrated that hypertension is accompanied by increased insulin-mediated VSMC growth due to sustained mitogen-activated protein kinase (MAPK) activity via a reduction of MAPK phosphatase-1 induction (3)(4)(5). Although VSMC proliferation and apoptosis are areas that are under intense investigation currently, very little is known about the role of prostaglandins (PGs) in the control of these processes, and even less is known about the biochemical pathways that regulate them.
Lipocalin-type prostaglandin D 2 synthase (L-PGDS) is responsible for the synthesis of PGD 2 from the common precursor PGH 2 . PGD 2 has been proven to induce sleep, inhibit platelet aggregation, inhibits nitric oxide release, induce vasodilation, and act as an allergic and inflammatory lipid mediator (6 -8). PGD 2 is also the precursor of 15d-PGJ 2 , a natural PPAR (ligand known to cause apoptosis) (9). L-PGDS is considered a dual functioning protein because it also acts as a lipophilic ligand-binding protein, binding and transporting retinoids, thyroids, and bile pigments (9). Depending upon its glycosylation or phosphorylation status, L-PGDS migrates anywhere from 20 to 29 kDa on SDS-PAGE.
Recent studies have linked L-PGDS to a variety of pathophysiological conditions in humans. For example, elevated serum L-PGDS levels after coronary angioplasty correlate with a decreased occurrence of restenosis (10); L-PGDS is secreted into, and accumulates in, the coronary circulation of angina patients (11,12); urinary L-PGDS excretion increases in the early stages of diabetic nephropathy (13); L-PGDS metabolism has been shown to be related to blood pressure and kidney injuries associated with hypertension (14); human L-PGDS mRNA is most intensely expressed in heart tissue, and the immunoreactivity of the enzyme is localized in myocardial cells, arterial endocardial cells, and the synthetic state of smooth muscle cells in arteriosclerotic plaques (12); and laminar shear stress stimulates endothelial cells to produce PGD 2 and 15d-PGJ 2 by up-regulating the expression of L-PGDS (15). It is felt that because PGD 2 is known as an anti-aggregatory prostanoid against human platelets, L-PGDS may be up-regulated to protect against platelet aggregation in atherosclerotic blood vessels (16).
Studies from this laboratory have demonstrated that L-PGDS induces apoptosis in both LLC-PK1 pig kidney epithelial and PC12 rat neuronal cells, presumably via the production of the PPAR␥ agonist, 15d-PGJ 2 (17,18). We have also deter-mined that phorbol ester-induced apoptosis is mediated by L-PGDS phosphorylation and activation by protein kinase C, and is accompanied by an inhibition of the PI3K/PKB antiapoptotic signaling pathways (19). In the present study, we report on the elevation of L-PGDS in VSMCs isolated from SHR and the stimulation of enhanced L-PGDS production upon cell differentiation. We demonstrate the differential effects of L-PGDS on serum-induced cell proliferation and apoptosis in VSMCs isolated from spontaneously hypertensive rats (SHR) versus normotensive Wistar-Kyoto (WKY) controls. We also examined the effects of L-PGDS on Akt and glycogen synthase kinase-3␤ (GSK-3␤) phosphorylation in response to insulin. We propose that L-PGDS is involved in maintaining a proper balance between cell proliferation and apoptosis in the hypertensive and hyperglycemic states.

EXPERIMENTAL PROCEDURES
Materials-Cell culture reagents, including fetal bovine serum, were purchased from Invitrogen. SDS/polyacrylamide gel electrophoresis and Western blot reagents were from Bio-Rad. Signal transduction antibodies were purchased from Cell Signaling Technology (Beverly, MA) and Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Antibody against L-PGDS was from Cayman Chemical (Ann Arbor, MI). Bicinchoninic acid protein assay reagent was purchased from Pierce. Western blots were visualized with enhanced chemiluminescence reagent purchased from Amersham Biosciences. Porcine insulin was a kind gift from the Eli Lilly Co. Type 1 collagenase was from Worthington. The caspase-3 activity apoptotic detection kit was purchased from R & D Systems (Minneapolis, MN). All other reagents were purchased from Sigma.
Cell Culture-VSMCs were isolated by collagenase digestion of the aortic media from male normotensive WKY and SHR rats with body weights between 200 and 220 g, as described in our recent publications (3)(4)(5). VSMCs prepared from these rats were not contaminated with fibroblasts or endothelial cells, as evidenced by a greater than 99% positive immunostaining of smooth muscle ␣-actin with fluorescein isothiocyanate-conjugated ␣-actin antibody. Subcultures of VSMCs were maintained in ␣-minimum essential medium containing 10% FBS and 1% antibiotic/antimycotic. Unless otherwise stated, cells were grown to 70 -80% confluency and studied at passages 4 -5 for all experiments.
Western Blotting-Culture plates were washed four times with icecold PBS followed by the addition of cell lysis buffer containing 50 mM HEPES, pH 7.6, 2.0 mM EDTA, 2.0 mM EGTA, 1.0% SDS, 1.0 mM benzamidine, 2.0 mM phenylmethylsulfonyl fluoride, and 10 g/ml each leupeptin, aprotinin, antipain, soybean trypsin inhibitor, and pepstatin A. When phosphorylation was detected, PBS and cell lysis buffer contained 2 mM sodium vanadate and 1 m microcystin at 4°C. The plates were scraped, and the cell lysate was sonicated and centrifuged at 2000 ϫ g for 5 min. Typically, 50 g of protein was mixed with Laemmli sample buffer containing 0.1% bromphenol blue, 1.0 M NaH 2 PO 4 , pH 7.0, 50% glycerol, and 10% SDS, boiled for 5 min, and loaded on an SDS-polyacrylamide gel. The separated proteins were transferred to polyvinylidene difluoride membrane and probed with the proper antibody followed by detection with enhanced chemiluminescence reagent and subsequent autoradiography. The intensity of the signal was quantitated by densitometric analysis of the autoradiograms.
Apoptotic Activity Assay-Apoptosis was quantitated as described previously (19) by the colorimetric measurement of caspase-3 activity as per the manufacturer's procedure. In addition, conformation of apoptosis was determined by TUNEL assay as described previously (19) using the ApopDetek Cell Death Assay Kit (Enzo, Farmingdale, NY).
Apoptotic Gene Expression by Gene Array-Cells were grown as described above in the presence or absence of L-PGDS (50 g/m) for 15 h, and total RNA was isolated using Trizol reagent (Invitrogen). Typically, 10 g of RNA was used as a template for [ 32 P]cDNA probe synthesis using [␣-32 P]dCTP (Amersham Biosciences). The gene array membrane (SuperArray, Frederick, MD) was prehybridized for 2 h at 68°C, and then denatured cDNA probe was added and hybridized for 15 h at 68°C. The membrane was washed twice with solution 1 (2ϫ SSC, 1% SDS) and twice with solution 2 (0.1ϫ SSC, 0.5% SDS) for 20 min each at 68°C with agitation. The wet membrane was wrapped with plastic wrap and exposed to x-ray film with an intensifying screen at Ϫ70°C.
Cell Proliferation and DNA Synthesis-Cell proliferation was assessed by counting cell numbers. After the specified incubation period, cells were washed twice with ice-cold PBS, harvested with trypsin, and centrifuged. Cell pellets were resuspended in ice-cold PBS and counted with a hemocytometer. Growth rate was determined by plotting cell numbers over time in semi-log scale and extrapolating the slope. DNA synthesis was assessed by [ 3 H]thymidine incorporation as described previously (4). Briefly, VSMCs from WKY and SHR were grown to 70% confluence (5 days in culture) as described above. After a 12-h pretreatment with either L-PGDS (50 g/ml) or buffer (0.1 M NaPO 4 , pH 7.4), the cells were treated for 15 min with insulin (100 nM) followed by [ 3 H]thymidine incorporation for 3 h. At the end of the incubation, the medium was removed, and the cell monolayers were washed sequentially with ice-cold PBS (three times) and once with ice-cold trichloroacetic acid (10%). The cells were solubilized by the addition of 1 ml of 0.1% SDS, 0.1 N NaOH. The solubilized extract (700 l) was added to 6 ml of scintillation fluid, and the incorporation of [ 3 H]thymidine into DNA, determined by liquid scintillation spectrometry, was expressed as dpm/mg of protein.
Protein Concentration Assay-Protein in cellular lysates was quantitated by the bicinchoninic acid method (20).
Statistics-Data are expressed as the mean Ϯ S.E. Analysis of variance and the Student's t test were used to compare the mean values between various treatments. A p value of Ͻ0.05 was considered statistically significant.

L-PGDS Expression Is Elevated in SHR and Increases upon
VSMC WKY Differentiation-Because L-PGDS metabolism appears to be associated with hypertension and vascular complications, we decided to look at L-PGDS expression in VSMCs isolated from WKY and SHR. Interestingly, L-PGDS levels were ϳ50% higher in SHR cells when compared with undifferentiated WKY cells (Fig. 1A, lane 3 versus lane 1). Furthermore, upon differentiation (day 10), L-PGDS expression increased 50% in WKY cells (Fig. 1A, lane 2 versus lane 1) and slightly in SHR cells (Fig. 1A, lane 4 versus lane 3). proliferation, we measured total cell numbers by cell counting and DNA synthesis by [ 3 H]thymidine incorporation in response to exogenously added L-PGDS. As seen in Fig. 2A, L-PGDS treatment had a slight inhibitory effect on WKY cell doubling. A 50% inhibition of growth, however, was observed in SHR cells after 5 days in culture with L-PGDS (Fig. 2B). Concomitantly, the 2-fold stimulation of DNA synthesis observed in the presence of serum, in both WKY and SHR, was completely inhibited in the presence of L-PGDS in SHR, but not WKY, VSMCs (Fig.  3). It is worth noting that the basal level of DNA synthesis was 3-fold higher in SHR as compared with WKY cells.
Effect of L-PGDS Exposure on Apoptosis-Recently, we observed the induction of apoptosis by L-PGDS in several cell lines (17,18). Because L-PGDS significantly inhibited the serum-induced proliferation of SHR VSMCs, we decided to examine the effect of L-PGDS on apoptosis in VSMCs isolated from both WKY and SHR rats. Fig. 4 demonstrates a 55% increase in the induction of caspase-3 activity by L-PGDS in serum-induced SHR VSMCs when compared with serum alone. Caspase-3 activity was not significantly altered by L-PGDS in WKY cells in the presence or absence of serum (Fig. 4). It is worth noting that the absolute level of apoptosis, as measured by caspase-3 activity, was nearly 2-fold higher in SHR when compared with WKY cells and that similar results were observed using the TUNEL assay to measure apoptosis (data not shown).
Furthermore, we examined the effect of L-PGDS exposure on the induction of known apoptotic genes using gene array hybridization. Exposure of WKY cells to L-PGDS for 15 h, in the presence of serum, had no effect on the induction of any of the apoptotic genes studied (Fig. 5A), whereas exposure of SHR cells to L-PGDS activated the expression of bax (coordinates C1 and D1), bcl-x (coordinates C2 and D2), and ei24 (coordinates C7 and D7), three apoptosis-related genes (Fig. 5B).
L-PGDS Pretreatment Inhibits the Insulin-induced Phosphorylation of Akt and GSK-3␤ and Induces Apoptosis in Insulintreated WKY VSMCs-The PI3K pathway has been shown to be involved in the apoptosis of VSMCs (21)(22)(23)(24). Previous work from this laboratory has linked both Akt and GSK-3␤ phosphorylation to L-PGDS-induced apoptosis (19). Insulin, a known inducer of Akt and GSK-3␤ phosphorylation, has been shown to stimulate proliferation and inhibit VSMC apoptosis (22). Be-  (Fig. 8A, lane 3 versus lane 2), whereas SHR cells pretreated with L-PGDS exhibited no inhibition of insulin-stimulated GSK-3␤ phosphorylation (Fig. 8A,  lane 6 versus lane 5). Both processes appear to be independent of the ERK pathway because insulin-stimulated ERK phosphorylation was unaffected by L-PGDS pretreatment in both WKY and SHR (Fig. 9).
Effect of Hyperglycemia on L-PGDS Protein Expression-Hyperglycemia, associated with increased VSMC proliferation and the inhibition of apoptosis (26,27), has been linked to hypertension and insulin resistance (28,29). Blood sugar control has been associated with urinary L-PGDS excretion in Type II diabetics (30). We therefore examined the effect of hyperglycemia on L-PGDS expression in VSMCs. Fig. 10 demonstrates the inhibition of L-PGDS expression in response to high glucose (20 mM) in WKY cells. Interestingly, the opposite was observed in SHR cells, where growth under hyperglycemic conditions actually increased L-PGDS expression 2-fold. DISCUSSION In this study, we examined L-PGDS expression in VSMCs isolated from normotensive and hypertensive rats and found a 50% elevation in L-PGDS expression in SHR compared with WKY controls (Fig. 1). Interestingly, patients with essential hypertension have a significant elevation in their serum L-PGDS levels (14). This SHR phenotype was maintained up through the sixth passage of our primary VSMC cultures, whereas subsequent passages had L-PGDS levels more comparable with WKY. We also examined the effect of exogenously added L-PGDS on cell proliferation and apoptosis and demon-strated the ability of LPGDS to significantly inhibit SHR VMSC proliferation (Figs. 2 and 3) and concomitantly stimulate SHR apoptosis (Fig. 4). Caspase-3 activity as well as the TUNEL assay (data not shown) were used to confirm apoptosis. In addition, gene array analysis confirmed the activation of apoptosis in SHR by the induction of bax, bcl-x, and ei24 (Fig.  5). We have confidence in the array technique because the negative bacterial plasmid controls (coordinates G1 and G2) as well as the positive actin and glyceraldehyde-3-phosphate dehydrogenase controls (coordinates G3 and G4 and G5-G8, E8, F8, respectively) all gave the predicted results (Fig. 5). These observations are also consistent with the increased apoptotic levels observed in target organs of SHR rats (31,32). It is noteworthy that L-PGDS expression increased in both SHR and WKY upon differentiation, a stage of development known to have increased levels of apoptosis.
Recent evidence suggests that arachidonic acid metabolites, namely prostaglandins, are involved in regulating cell proliferation. Inhibition of 85-kDa phospholipase A 2 , the enzyme responsible for hydrolyzing membrane phospholipids into arachidonic acid, resulted in a dose-dependent inhibition of VSMC proliferation (33). 15d-PGJ 2 , which is quickly formed from PGD 2 , is known to inhibit basic fibroblast growth factor-induced DNA synthesis in rat VSMCs (34). 15d-PGJ 2 also inhibits platelet-derived growth factor-directed migration (34) and induces G 1 arrest and differentiation (35) in VSMCs. Similar effects have been observed with the synthetic PPAR␥ activator, troglitazone, which inhibits VSMC proliferation (36), decreases the intimal and medial thickness of carotid arteries in humans (37), and inhibit the development of atherosclerosis (38). It is our hypothesis that L-PGDS functions to stimulate the production of these PGs and thereby inhibits cell proliferation and FIG. 5. Effect of L-PGDS on apoptotic gene expression. Total RNA was isolated from VSMCs cultured as described under "Experimental Procedures" in the presence or absence of (50 g/ml) L-PGDS for 18 h. 10 g of RNA was used as a template for [ 32 P]cDNA probe synthesis. The gene array membranes (A, WKY; B, SHR) were hybridized for 15 h at 68°C and washed as described under "Experimental Procedures." The membranes were exposed to film at Ϫ70°C for several days and the autoradiogram scanned with SigmaGel software. Apoptotic gene coordinates: bax, C1/D1; bcl-x, C2/D2; ei24 (pig8), C7/D7. stimulates apoptosis in hypertension and diabetes.
The role of PGs in VSMC proliferation has also been demonstrated directly in SHR VSMCs. For example, the addition of indomethacin, a cyclooxygenase inhibitor, leads to increased proliferation of VSMCs in SHR rats (39). In addition, the inducible gene responsible for NO synthesis in VSMCs, nitric oxide synthase, is inhibited by both PGD 2 and 15d-PGJ 2 but not by PGE 2 , PGI 2 , or PGF 2 (40). In addition, the effect of retinoic acid metabolism on cell growth has been investigated for its potential as an anticancer agent because of its ability to inhibit cell proliferation, migration, and differentiation of VSMCs (41). In human coronary artery VSMCs, the antiproliferative effects of 15d-PGJ 2 were enhanced 4-fold with the addition of 9-cis-retinoic acid, a retinoid X receptor ligand (42). Although this study (42) does not address the apoptotic state of the cells, it does demonstrate the effectiveness of the combination of 15d-PGJ 2 and retinoic acid in terms of the inhibition of VSMC proliferation. It is worth noting that in addition to its function as the enzyme responsible for the eventual synthesis of 15d-PGJ 2 , L-PGDS is also a retinoid transporter. Several growth factors have been shown to alter the balance between VSMC proliferation and apoptosis. For example, insulin-like growth factor (IGF-1) has been shown to inhibit apoptosis and promote cell proliferation (43). Bai et al. (44) have shown that apoptosis is prevented by IGF-1 via the PI3K signaling pathway, which inactivates the proapoptotic gene, Bad, by phosphorylation presumably via Akt. In addition, protein kinase C and p38 MAPK activation have been reported to cause an increase in PG formation (45), and elevated protein kinase C levels have also been reported in VSMCs of diabetic rats (46). Similarly, we have observed the decreased activation of PI3K FIG. 8. Effect of L-PGDS on insulin-stimulated GSK-3␤ phosphorylation. VSMCs were cultured as described under "Experimental Procedures" in medium containing 10% FBS in the presence or absence of L-PGDS (50 g/ml) for 2 h. Where indicated, insulin (100 nM) was added for 10 min. Protein lysate (50 g) was separated by SDS-PAGE on a 12% gel, transferred to a PVDF membrane, and probed for the 46-kDa phospho-GSK-3␤ (pGSK-3␤) and GSK-3␤ (A). Panel B represents the phosphorylated GSK-3␤ intensity corrected for GSK-3␤ expression. Values are the mean Ϯ S.E. of four experiments. *, p Ͻ 0.05 when compared with insulin-stimulated cells.
FIG. 9. Effect of L-PGDS on insulin-stimulated ERK phosphorylation. VSMCs were cultured as described under "Experimental Procedures" in medium containing 10% FBS in the presence or absence of L-PGDS (50 g/ml) for 2 h. Where indicated, insulin (100 nM) was added for 10 min. Protein lysate (50 g) was separated by SDS-PAGE on a 12% gel. Proteins were transferred to a PVDF membrane and probed for the 42-kDa phospho-ERK (pERK) and ERK (A). Panel B represents the phosphorylated ERK intensity corrected for ERK expression. Values are the mean Ϯ S.E. of three experiments. There was no significant difference in ERK phosphorylation between WKY and SHR. (3) and increased p38 MAPK activation and phosphorylation in VSMCs isolated from SHR rats (47). Our present results suggest that when VSMCs are in a hyperproliferative state, such as in serum-induced SHR or upon differentiation, L-PGDS expression is elevated to inhibit cell proliferation and stimulate cellular apoptosis. It is only when proliferation pathways are active that L-PGDS can induce apoptosis and inhibit cell proliferation. This is consistent with our previous findings in other cell lines, where L-PGDS-induced apoptosis is more pronounced in the presence of serum (19). In addition, L-PGDS pretreatment appears to inhibit insulin-stimulated proliferation by blocking any further Akt/GSK-3␤ phosphorylation in wild-type VSMCs (Figs. 7 and 8), inducing a beneficial "insulin resistance" environment. L-PGDS may act as a moderator for the effects of insulin on cell proliferation and apoptosis, and it may help control WKY proliferation, despite the presence of insulin. This observation is made in differentiated WKY VSMCs, which express significant levels of L-PGDS. It appears that these L-PGDS effects are independent of the ERK pathway, because there was no effect on ERK phosphorylation. Future studies will investigate the c-Jun N-terminal kinase (JNK) and p38/MAPK pathways in more depth.
Hyperglycemia, another condition favoring hyperproliferation, has been shown to decrease apoptosis of wild-type VSMCs (27). Our results involving hyperglycemia (Fig. 10) also support the proapoptotic role for L-PGDS under growth-stimulated conditions. When WKY cells were incubated in high glucose, L-PGDS expression decreased, consistent with the decrease in apoptosis, and increased cell proliferation observed under hyperglycemic conditions. SHR VSMCs actually increased L-PGDS expression (Fig. 10), possibly to help control the hyperproliferation that already exists in this cell line.
In the present study, we report the elevation of L-PGDS in VSMCs isolated from SHR and its further increase upon VSMC differentiation. We demonstrate differential effects of L-PGDS on serum-and insulin-induced cell proliferation and apoptosis in VSMCs isolated from SHR versus normotensive controls. In addition, we examined the effects of L-PGDS on Akt and GSK-3␤ phosphorylation. We hypothesize that increased L-PGDS expression and secretion is a cellular mechanism to inhibit cell proliferation and stimulate apoptosis in the hypertensive and hyperglycemic pathogenic states. Therefore, we believe that L-PGDS represents a potential therapeutic site in the treatment of atherosclerosis.