Inhibition of cell cycle progression and migration of vascular smooth muscle cells by prostaglandin D2 synthase: resistance in diabetic Goto-Kakizaki rats

Louis Ragolia,1,2 Thomas Palaia,1 Tara B. Koutrouby,1 and John K. Maesaka1,2

1Vascular Biology Laboratory, Winthrop-University Hospital, Mineola 11501; and the 2Stony Brook University School of Medicine, Stony Brook, New York 11794

Submitted 11 May 2004 ; accepted in final form 1 July 2004


    ABSTRACT
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The regulation of vascular smooth muscle cell (VSMC) proliferation, migration, and apoptosis plays a clear role in the atherosclerotic process. Recently, we reported on the inhibition of the exaggerated growth phenotype of VSMCs isolated from hypertensive rats by lipocalin-type prostaglandin D2 synthase (L-PGDS). In the present study, we report the differential effects of L-PGDS on VSMC cell cycle progression, migration, and apoptosis in wild-type VSMCs vs. those from a type 2 diabetic model. In wild-type VSMCs, exogenously added L-PGDS delayed serum-induced cell cycle progression from the G1 to S phase, as determined by gene array analysis and the decreased protein expressions of cyclin-dependent kinase-2, p21Cip1, and cyclin D1. Cyclin D3 protein expression was unaffected by L-PGDS, although its gene expression was stimulated by L-PGDS in wild-type cells. In addition, platelet-derived growth factor-induced VSMC migration was inhibited by L-PGDS in wild-type cells. Type 2 diabetic VSMCs, however, were resistant to the L-PGDS effects on cell cycle progression and migration. L-PGDS did suppress the hyperproliferation of diabetic cells, albeit through a different mechanism, presumably involving the 2.5-fold increase in apoptosis and the concomitant 10-fold increase of L-PGDS uptake we observed in these cells. We propose that in wild-type VSMCs, L-PGDS retards cell cycle progression and migration, precluding hyperplasia of the tunica media, and that diabetic cells appear resistant to the inhibitory effects of L-PGDS, which consequently may help explain the increased atherosclerosis observed in diabetes.

apoptosis; atherosclerosis; insulin resistance


THE REGULATION of vascular smooth muscle cell (VSMC) proliferation, apoptosis, and migration is vital to the pathogenesis of atherosclerosis, the primary cause of mortality in persons with diabetes (31). Recently, we demonstrated that lipocalin-type prostaglandin D2 synthase (L-PGDS) suppresses the exaggerated cell proliferation observed in spontaneously hypertensive VSMCs by stimulating apoptosis (43). In addition, we determined that phorbol ester-induced apoptosis is mediated by L-PGDS phosphorylation and is accompanied by the inhibition of the phosphatidylinositol 3-kinase (PI3-K) and protein kinase B (Akt) antiapoptotic signaling pathways (42). While it is clear that VSMC proliferation, apoptosis, and migration are all important factors influencing cardiovascular disease, the precise mechanisms regulating these processes remain uncertain.

L-PGDS, a unique lipocalin responsible for both the synthesis of prostaglandin (PG) D2 and the transport of retinoids (50), has also been associated with a variety of cardiovascular conditions in humans. For example, a decreased occurrence of restenosis after coronary angioplasty correlates with elevated serum L-PGDS levels (23); urinary L-PGDS excretion increases in the early stages of diabetic nephropathy (19); L-PGDS is accumulated in the coronary circulation of angina patients (13, 51); L-PGDS metabolism has been linked to hypertension (20); 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 (13); L-PGDS is a genetic loci that controls VEGF-induced angiogenesis (44); and finally, laminar shear stress stimulates endothelial cells to produce PGD2 by upregulating L-PGDS expression (49). Because PGD2 has been proven to inhibit platelet aggregation, inhibit nitric oxide release, induce vasodilatation, and act as an inflammatory lipid mediator (6, 33, 34), it is felt that L-PGDS may be upregulated to protect against platelet aggregation in atherosclerotic blood vessels (52).

PGD2 is also the precursor of 15-deoxy-{Delta}12,14-prostaglandin J2 (15-dPGJ2) a natural ligand for peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) known to cause apoptosis (51), induce G1 arrest (36), and inhibit the migration of VSMCs (32). Thiazolidinediones (TZDs), which are synthetic ligands for PPAR-{gamma}, have been shown to inhibit VSMC growth and intimal hyperplasia (27) and decrease G1 cyclin levels and inhibit cell cycle progression independent of p21Cip1 and p27Kip1 (22). In addition, other beneficial cardiovascular effects have been attributed to TZDs such as blocking atherosclerosis in apolipoprotein E-knockout mice (8) and inhibiting VSMC migration (10).

Goto-Kakizaki (GK) rats, a nonobese model for type 2 diabetes, were isolated by the excessive inbreeding of Wistar-Kyoto (WKY) rats that spontaneously develop type 2 diabetes (15). Rats with this genetic pedigree are relevant to human type 2 diabetes due to the defects in glucose-stimulated insulin secretion, peripheral insulin resistance, hyperglycemia (9.3 ± 1.1 vs. 6.9 ± 0.2 mmol/l in controls), hypertension (183 ± 14 vs. 144 ± 6 mmHg in controls), and hyperinsulinemia (8.1 ± 1.1 vs. 3.0 ± 0.7 ng/ml in controls), all observed as early as 4 wk after birth (9, 15, 24). Previously, we concluded that the hyperglycemia associated with diabetes is accompanied by a marked impairment of myosin-bound phosphatase (MBP) activation by insulin, using the GK model system (45). MBP inhibition in GK cells paralleled an enhanced VMSC contraction, which resulted from increased myosin light chain20 phosphorylation (2, 45). In the present study, we report on the differential effects of L-PGDS on VSMC cell cycle progression, migration, and apoptosis in wild-type VSMCs vs. those from a type 2 diabetic model. We propose that L-PGDS retards cell cycle progression and migration of wild-type VSMCs, precluding hyperplasia of the tunica media, and that diabetic cells appear resistant to the inhibitory effects of L-PGDS, which consequently may help explain the increased atherosclerosis observed in diabetes.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Cell culture reagents, including FBS and platelet-derived growth factor (PDGF)-BB, were all purchased from Life Technologies (Grand Island, NY). SDS-PAGE and Western blot reagents were from Bio-Rad (Hercules, CA). Cell cycle gene arrays (cat. no. MM-001) were purchased from SuperArray (Frederick, MD). [3H]thymidine was purchased from Dupont/New England Nuclear (Boston, MA). Signal transduction antibodies were purchased from Cell Signaling Technology (Beverly, MA) and Santa Cruz Biotechnology (Santa Cruz, CA). Bicinchoninic acid protein assay reagent was purchased from Pierce (Rockford, IL). Western blots were visualized with enhanced chemiluminescence reagent purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Type-1 collagenase was from Worthington Biochemical (Freehold, NJ). All other reagents were purchased from Sigma Chemical (St. Louis, MO).

Cell culture. VSMCs were isolated by collagenase digestion of the aortic media from male WKY rats and GK diabetic rats with body weights between 200 and 220 g, as described previously (25, 43). VSMCs prepared from these rats were not contaminated with fibroblasts or endothelial cells as evidenced by a >99% positive immunostaining of smooth muscle {alpha}-actin with fluorescein isothiocyanate-conjugated {alpha}-actin antibody. Subcultures of VSMCs were maintained in {alpha}-MEM containing 10% FBS, and 1% antibiotic-antimycotic. Cells were synchronized in G0 by incubation in serum-free medium for 24 h. Cells were grown to confluency and studied at passages 5–6 for all experiments.

Western blotting. Cells were lysed as previously described (5). When phosphorylation was detected, PBS and cell lysis buffer contained 2 mM sodium vanadate and 1 µm microcystin at 4°C. Typically, 50 µg of protein were mixed with Laemmli sample buffer containing 0.1% bromophenol blue, 1.0 M NaH2PO4, 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 and normalized against {beta}-actin, or in the case of phosphorylations, the nonphosphorylated form of the protein.

Apoptotic activity assay. Apoptosis was quantitated as previously described by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) (43) using the ApopDetek Cell Death Assay Kit (Enzo, Farmingdale, NY). Conformation of apoptosis was determined by the colorimetric measurement of caspase-3 activity (R&D, Minneapolis, MN) per the manufacturer's instructions.

Cell proliferation and DNA synthesis. Cell proliferation was assessed by counting cell number. 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 number over time in semilog scale and extrapolating the slope. DNA synthesis was assessed by [3H]thymidine incorporation as described previously (5). Briefly, VSMCs from WKY and GK were grown to 70% confluence (5 days in culture) as described above. After a 15-h treatment with either L-PGDS (50 µg/ml) or buffer (0.1 M NaPO4, pH 7.4), the cells were exposed to [3H]thymidine for 3 h. At the end of the incubation, the medium was removed and the cell monolayers were washed sequentially with ice-cold PBS (3x) and once with ice-cold TCA (10%). The cells were solubilized by the addition of 1 ml of 0.1% SDS-0.1 N NaOH. The solubilized extract (700 µl) was added to 6 ml of scintillation fluid, and the incorporation of [3H]thymidine into DNA was determined by liquid scintillation spectrometry and expressed as disintegrations per minute (dpm) per milligram of protein.

Cell migration assay. Migration assays were performed using 24-well cell culture inserts with 8.0-µm polyethylene terephthalate cyclopore membranes (Falcon) as detailed in Lundberg et al. (29). The underside of the membrane was coated with 10 µl of rat tail collagen type I (50 µg/ml) for 18–20 h, washed, and air-dried before each experiment. Serum-starved VSMCs were trypsinized and resuspended in {alpha}-MEM. Then 2 x 104 VSMCs/250 µl were loaded into the cell culture inserts. The inserts were then added to the wells of 24-well plates, which were filled with PDGF-BB diluted in {alpha}-MEM with 0.1% BSA, and where indicated, L-PGDS was also added to this medium below the inserts. The chambers were then incubated at 37°C for 5 h to allow for cell migration. Afterward, cells were completely removed from the upper side of the membrane with a cotton swab and the remaining migrated cells fixed and stained with Diff-Quik solution (Dade Behring, Newark, DE). Results are reported as means ± SE of five different fields, from three experiments, counted at x200 magnification.

Fluorescent labeling of L-PGDS. L-PGDS was fluorescently labeled using the Alexa Fluor 488 labeling kit (Molecular Probes, Eugene, OR) as per the manufacturer's instructions.

Assay of PGD2 levels. PGD2 levels were determined using a PGD2 methyloxylamine (MOX) hydrochloride kit (Cayman Chemical, Ann Arbor, MI). This assay is based on the conversion of PGD2 into a stable MOX derivative. Briefly, 50 µl of either culture medium or cell lysate were mixed with an equal volume of MOX reagent and processed as per the manufacturer's instructions.

Protein concentration assay. Protein in cellular lysates was quantitated by the bicinchoninic acid method (47).

Statistics. Unless noted, all data are expressed as means ± SE of at least three experiments performed in duplicate. ANOVA and the Student's t-test were used to compare the mean values between various treatments. A P value of <0.05, or lower, was considered statistically significant.


    RESULTS
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
L-PGDS stimulates apoptosis and inhibits the hyperproliferation of GK VSMCs. To determine the effects of L-PGDS on VSMC proliferation, we counted total cell number after the exposure of VSMCs to exogenously added L-PGDS. As seen in Fig. 1A, L-PGDS treatment (50 µg/ml) had a slight inhibitory effect on WKY cell doubling after 5 days in culture but caused nearly a 50% inhibition of GK growth after 3 days in culture (Fig. 1B). Similar results were obtained measuring DNA synthesis by [3H]thymidine incorporation (Fig. 1C).



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Fig. 1. Effect of lipocalin-type prostaglandin D2 synthase (L-PGDS) on cell proliferation and DNA synthesis. Vascular smooth muscle cells (VSMCs) were cultured as described in EXPERIMENTAL PROCEDURES in medium containing 10% FBS in the presence or absence of L-PGDS (50 µg/ml). Cells, either Wistar-Kyoto (WKY) control (A) or Goto-Kakizaki (GK; B), were plated at a density of 1 x 104 cells/ml in 35-mm dishes and, after the indicated time, counted using a hemocytometer. C: a representation of DNA synthesis. Cells at ~70% confluency were treated with L-PGDS (50 µg/ml) for 15 h followed by [3H]thymidine (1 µCi/ml) for an additional 3 h. Cells were washed 3 times, and the radioactivity was counted in a liquid spectrophotometer. Results are means ± SE of at least 3 experiments, each performed in duplicate. *P < 0.01 compared with untreated cells.

 
Recently, we observed the induction of apoptosis by exogenously added L-PGDS in several cell lines such as proximal tubule cells, neuronal cells, and VSMCs isolated from spontaneously hypertensive rats (30, 41, 43). We decided to examine the apoptotic effect of L-PGDS on VSMCs isolated from diabetic GK rats. Figure 2 demonstrates a 2.5-fold increase in TUNEL-positive cells after exposure of GK VSMCs to L-PGDS and only a slight stimulation by L-PGDS in WKY VSMCs (Fig. 2A). It is worth noting that the basal levels of apoptosis were nearly twofold higher in GK cells and that these results were confirmed by caspase-3 activity (Fig. 2B).



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Fig. 2. Effect of L-PGDS on apoptosis. VSMCs, isolated from WKY and GK rats, were cultured as described in EXPERIMENTAL PROCEDURES. At ~100% confluency the cells, where indicated, were treated with L-PGDS (50 µg/ml) for 18 h, and the apoptotic index was calculated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay (A) or caspase-3 assay (B). Results are means ± SE of 3 different experiments each performed in duplicate. *P < 0.05 vs. GK control.

 
Increased transport of L-PGDS into hyperproliferating GK VSMCs. The transport of L-PGDS into VSMCs was measured using fluorescently labeled L-PGDS and monitoring the in situ cellular appearance of the protein after its addition to the culture medium. As seen in Fig. 3, the transport of fluorescently labeled L-PGDS into GK cells was tenfold higher than transport into wild-type WKY cells after 5 h of incubation (Fig. 3B). Figure 3A represents the corresponding phase-contrast images of both cell lines.



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Fig. 3. Differential transport of L-PGDS into VSMCs. VSMCs were cultured as described in EXPERIMENTAL PROCEDURES in the presence or absence of fluorescently (AlexaFluor 488) labeled L-PGDS (50 µg/ml) for 5 h. Cells were washed 4 times in PBS and photographed with a Sony 3-CCD camera at x10 magnification under either phase-contrast (A) or UV (B) conditions.

 
L-PGDS pretreatment inhibits serum-induced expression of cell cycle proteins. To track cell cycle progression, the expressions of various protein markers were measured by immunoblot analysis. As seen in Fig. 4A, serum exposure stimulated the expression of cyclin-dependent kinase 2 (cdk2), cyclin D1, and cyclin D3 in WKY cells (Fig. 4A, lane 2 vs. lane 1). When serum induction was performed in the presence of L-PGDS, protein expressions of cdk2 and cyclin D1 were inhibited in wild-type WKY cells (Fig. 4A, lane 3 vs. lane 2) but remained elevated in diabetic GK cells (Fig. 4A, lane 6 vs. lane 5). Cyclin D3 expression was unchanged in both cell lines. Noteworthy are the serum-starved expressions of cyclin D1 and cyclin D3, which were elevated in GK cells (Fig. 4A, lane 4).



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Fig. 4. Effect of L-PGDS on serum-stimulated cell cycle protein expressions. VSMCs were cultured as described in EXPERIMENTAL PROCEDURES and serum starved for 24 h in {alpha}-MEM containing 1.0% ABS. Cells were then switched to medium containing 10% FBS in the presence or absence of L-PGDS (50 µg/ml) for 24 h. Protein lysates (50 µg) were separated by SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane, and probed with the appropriate antibodies. Representative autoradiograms are shown. Rb, retinoblastoma; pRb, phosphorylated Rb; GSK3{beta}, glycogen synthase kinase-3{beta}; pGSK3{beta}, phosphorylated GSK3{beta}; Akt, protein kinase B; pAkt, phosphorylated Akt; cdk2, cyclin-dependent kinase 2.

 
The effect of L-PGDS on the protein expression of cyclin-dependent kinase inhibitors was also examined in both wild-type and diabetic VSMCs. In both cell lines serum exposure stimulated the expression of p21Cip1 (Fig. 4B, WKY, compare lane 2 vs. lane 1; and GK, lane 5 vs. lane 4). In the presence of L-PGDS, the serum-induced protein expression of p21Cip1 was inhibited in WKY cells but remained elevated in diabetic GK cells (Fig. 4B, WKY, lane 3 vs. lane 2; and GK, lane 6 vs. lane 5). Similar L-PGDS resistance was obtained with p27Kip1. In WKY cells, serum exposure inhibited the expression of p27Kip1 (Fig. 4B, lane 2 vs. lane 1); however, serum treatment in the presence of L-PGDS reversed this inhibition (Fig. 4B, lane 3 vs. lane 2). There was no effect of L-PGDS on serum-induced p27Kip1 expression in the GK diabetic VSMCs, which had elevated p27Kip1 protein levels even under serum-free conditions.

L-PGDS inhibits serum-induced phosphorylation of retinoblastoma, Akt, and glycogen synthase kinase-3{beta}. The PI3-K pathway has been shown to be involved in the apoptosis of VSMCs (25, 39, 48, 54). Previous work from this laboratory has linked both Akt and glycogen synthase kinase-3{beta} (GSK-3{beta}) phosphorylation to L-PGDS-induced apoptosis in spontaneously hypertensive rats (43). Serum, a known inducer of retinoblastoma protein (Rb), Akt, and GSK-3{beta} phosphorylation, has been shown to stimulate proliferation and inhibit apoptosis of VSMCs (25). We decided to examine the effects of L-PGDS pretreatment on serum-stimulated Rb, Akt, and GSK-3{beta} phosphorylations. As seen in Fig. 4C, there is an approximate two- to threefold induction in the phosphorylation of Rb, Akt, and GSK-3{beta} in wild-type WKY VSMCs after serum induction (Fig. 4C, lanes 2 vs. lanes 1). In GK cells, serum-stimulated Akt and GSK-3{beta} phosphorylations are minimal due to high basal levels of phosphorylation (Fig. 4C, lane 5 vs. lane 4). Rb phosphorylation is, however, stimulated by serum in GK VSMCs. When cells were pretreated with L-PGDS, serum-stimulated phosphorylations of Akt, GSK-3{beta}, and Rb were all inhibited in wild-type WKY (Fig. 4C, lane 3 vs. lane 2) but unaffected in the hyperphosphorylated GK cells (Fig. 4C, lane 6 vs. lane 5). We believe that the increased basal levels of Akt phosphorylation observed in GK VSMCs are possible even with an accompanying increase in apoptosis due to the huge increase in the net amount of proliferation. Furthermore, it is quite possible that an alternative apoptotic pathway, independent of Akt, may be involved as suggested by Craddock et al. (11).

We believe that the hyperproliferation of GK VSMCs is accompanied by a concomitant increase in apoptosis. The net amount of proliferation, however, overshadows the apoptosis and is reflected in the increased basal levels of Akt phosphorylation observed. Furthermore, it is quite possible that an alternative apoptotic pathway, independent of Akt, may be involved. In fact, Craddock et al. has demonstrated the dissociation of cell proliferation/apoptosis from Akt phosphorylation.

L-PGDS pretreatment inhibits PDGF-induced VSMC migration. Migration of VSMCs, especially in response to PPAR-{gamma} ligands, plays an important role in the pathogenesis of cardiovascular disease in diabetes (21, 32). Because L-PGDS is responsible for the eventual synthesis of the PPAR-{gamma} ligand 15-dPGJ2, we decided to examine the effect of L-PGDS on PDGF-stimulated VSMC migration in both WKY and GK cells. A two- to fourfold increase in PDGF-induced migration was observed in GK and WKY cells, respectively (Fig. 5a, panel B vs. panel A). Interestingly, when incubated together, L-PGDS inhibited the migration of WKY VSMCs but had no effect on the migration of GK VSMCs (Fig. 5a, panel D vs. panel B). Figure 5b is a graphical representation of the results.



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Fig. 5. Effect of L-PGDS on platelet-derived growth factor (PDGF)-induced VMSC migration. VSMCs isolated from WKY and GK rats were cultured as described in EXPERIMENTAL PROCEDURES and loaded into collagen-coated 24-well cell culture inserts as described above. a: Actual fields at x200 magnification under conditions of 5 mM glucose. b: Graphical representation of means ± SE of 5 different fields from 3 experiments performed at 5 mM glucose and counted at x200 magnification. *P < 0.05 compared with respective control cells.

 
L-PGDS expression is elevated in GK and increases further under hyperglycemic conditions. Hyperglycemia is associated with increased VSMC proliferation (17), the inhibition of apoptosis (1), and insulin resistance (31). In addition, blood sugar control has been associated with urinary L-PGDS excretion in type II diabetics (18). Because L-PGDS metabolism appears to be associated with diabetes and is associated with the balance of VSMC apoptosis and proliferation, we decided to examine L-PGDS expression in VSMCs isolated from WKY and GK. Interestingly, L-PGDS levels were approximately twofold higher in GK cells compared with WKY cells (Fig. 6A, lane 3 vs. lane 1) under normal conditions (5 mM glucose). Furthermore, under hyperglycemic conditions (20 mM glucose), L-PGDS expression increased twofold over basal levels in GK cells (Fig. 6A, lane 4 vs. lane 3), while actually decreasing slightly in WKY cells (Fig. 6A, lane 2 vs. lane 1). Figure 6B represents the quantitation of L-PGDS intensity corrected for {alpha}-actin expression and demonstrates the fourfold increase in L-PGDS expression in GK vs. WKY, under hyperglycemic conditions (Fig. 6A, lane 4 vs. lane 2).



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Fig. 6. Hyperglycemic conditions increase L-PGDS protein expression in GK cells. VSMCs, isolated from WKY and GK rats, were cultured as described in EXPERIMENTAL PROCEDURES under either normal (5 mM glucose) or hyperglycemic (20 mM glucose) conditions. Proteins were extracted after 10 days of culturing, and 50 µg were separated by SDS-PAGE on a 12% gel. Proteins were transferred to a PVDF membrane and probed with both L-PGDS and actin antibodies (A). B: corrected L-PGDS expression normalized to actin expression. Results are means ± SE of 4 experiments. P < 0.05 compared with control WKY cells (*) or GK control cells (**).

 
PGD2 levels in VSMC lysates vs. the culture medium. PGD2 is the precursor of the known apoptotic inducer, 15-dPGJ2. Therefore, the intra- and extracellular levels of PGD2 were determined by ELISA in both wild-type and diabetic VSMCs. In the WKY culture medium, the level of PGD2 was 25 pg/ml, approximately twofold higher than the 14 pg/ml measured in the GK culture medium. In the harvested cell lysates, the converse was true. GK cells had a PGD2 level over fourfold higher than WKY (45 pg/ml compared with 10 pg/ml), respectively (Fig. 7).



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Fig. 7. PGD2 levels in cell medium and lysates. VSMCs, isolated from WKY and GK rats, were cultured as described in EXPERIMENTAL PROCEDURES. At ~100% confluency, the PGD2 concentration of both the culture medium and the cell lysate was determined by the conversion of PGD2 into a stable methyloxylamine product. Results are means ± SE of 3 experiments performed in duplicate and expressed as pg of PGD2/ml. P < 0.05 compared with WKY medium (*) or lysate (**).

 

    DISCUSSION
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Atherosclerosis is a gradual and complex process involving cross talk between the endothelium, macrophages, and VSMCs. Figure 8 categorizes the progression of atherosclerosis into three different stages and demonstrates the role of VSMCs in this process. Modification of the key cell types is accomplished by a variety of growth factors, cytokines, prostaglandins, and cholesterol. In this study, we have examined the effects of exogenously added L-PGDS on VSMC proliferation, apoptosis, cell cycle progression, and migration, as each contributes to the overall atherosclerotic process. We report the differential effects L-PGDS exhibits on each of these processes in wild-type WKY VSMCs vs. diabetic GK cells.



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Fig. 8. Progression of atherosclerosis. Three stages of atherosclerosis are presented with key cell types involved. LDL, low-density lipoprotein; oxLDL, oxidized LDL; VEC, vascular endothelial cell.

 
L-PGDS inhibits hyperproliferation of VSMCs. In diabetic GK cells, we demonstrated the ability of L-PGDS to significantly inhibit the hyperproliferation of cells in culture (Fig. 1) and concomitantly stimulate apoptosis (Fig. 2). These data are consistent with our previous findings wherein L-PGDS stimulated apoptosis and suppressed the exaggerated cell growth observed in VSMCs isolated from spontaneously hypertensive rats (43). The fact that L-PGDS induces apoptosis in GK cells to a greater extent than in WKY may be explained by the increased transport of L-PGDS into diabetic VSMCs (Fig. 3) or by differences between intracellular and culture medium PGD2 levels (Fig. 7). Also, the inhibition of L-PGDS on nedd8 and cullin3 gene expression (data not shown) might tilt the balance toward apoptosis because these genes have been shown to be involved in the regulation of mammalian cell growth (28, 37, 38). Alternatively, GK cells may be in an altered stage of development, as is evidenced by their hyperproliferation and elevated serum-free cyclin D1 levels (Fig. 4), and therefore more susceptible to the initiation of apoptosis. Furthermore, it has been shown that wild-type VSMCs cultured under hyperglycemic conditions will increase cell proliferation (14, 16) and decrease apoptosis through a protein kinase C (PKC)-dependent pathway (17). These data correlate well with our previous results demonstrating that phorbol ester-induced apoptosis was mediated by L-PGDS phosphorylation and activation by PKC (42) and also a current observation that there is decreased L-PGDS protein expression in wild-type VSMCs cultured under hyperglycemic conditions (unpublished results).

L-PGDS did stimulate apoptosis and inhibit the excessive growth of GK cells in culture. It is possible that L-PGDS controls wild-type VSMC growth by regulating cell cycle gene expression, and it is only under instances of uncontrollable growth, such as diabetes and hypertension, that VSMCs become resistant to the cell cycle effects of L-PGDS. Under these circumstances L-PGDS is transported into the cell, and excessive VSMC proliferation is controlled by the triggering of apoptosis. In fact, we have observed increased L-PGDS expression in VSMCs isolated from both hypertensive (43) and diabetic GK rats (Fig. 6). Furthermore, Chen and Gardner (7) have shown that under quiescent conditions, retinoids, which are known to be transported by L-PGDS, activate mitogenesis, whereas in the presence of growth stimulation, they actually suppress mitogenesis of VSMCs. Finally, Perlman et al. (40) have demonstrated the separation of apoptosis and cell cycle regulation by proving that Bax gene expression is independent of cell cycle proteins.

L-PGDS inhibits cell cycle progression. Immunoblot analysis of cell cycle proteins clearly demonstrates the regulatory role of L-PGDS in cell cycle progression and the resistance of diabetic GK cells. Expression of cyclin D1, cyclin D3, and cdk2 allows VSMCs to progress along the cell cycle. In the case of cyclin D1 and cdk2, L-PGDS was able to inhibit serum-induced protein expression in wild-type WKY VSMCs but failed to do so in diabetic GK cells (Fig. 4A). There were no observed effects of L-PGDS on either cyclin D3 or p27Kip1 protein expressions (Fig. 4, A and B), although there were alterations of their gene expressions in WKY cells (data not shown). Posttranscriptional regulation, which has been demonstrated to be involved in VSMC cell cycle regulation (46), probably plays a role here. The serum-induced protein expression of p21Cip1 was inhibited by L-PGDS in WKY cells and not GK cells (Fig. 4B), implicating this cyclin-dependent kinase inhibitor. Wakino et al. (51a) have shown that PPAR-{gamma} ligands regulate p21Cip1 at a posttranslational level by blocking PKC signaling and accelerating p21Cip1 turnover, and Zhang et al. (55) demonstrated that the overexpression of p21Cip1 in human VSMCs caused the inhibition of cell proliferation through apoptosis. These findings are in agreement with our previous findings linking L-PGDS-induced apoptosis to PKC (42), and our current p21Cip1 protein expression data (Fig. 4B). The fact that wild-type VSMC proliferation is only slightly decreased by L-PGDS (Fig. 1A, days 1–5), while the serum-induced cell cycle progression is significantly inhibited (Fig. 4), is probably due to the difference in cell confluency between the two types of experiments. Cell cycle protein phosphorylations were performed on fully confluent cells; when wild-type cells were approaching this stage, there was a significant inhibition of proliferation observed (Fig. 1A, day 5).

Cellular proliferation and migration are regulated in a coordinated manner by the p27Kip1/cdk/phosphorylated Rb pathway (12), and increased VSMC migration is believed to be an early atherosclerotic event (53). Inhibition of the serum-stimulated phosphorylations of Akt, GSK-3{beta}, and Rb by L-PGDS in WKY cells provides a solid link to these pathways, which is flawed in GK diabetic cells (Fig. 4C). Furthermore, the naturally occurring PPAR-{gamma} ligand and ultimate L-PGDS end product, 15-dPGJ2, has been shown to inhibit basic fibroblast growth factor-induced DNA synthesis and PDGF-directed migration (26) and induce G1 arrest and differentiation in rat VSMCs (36). Similarly, TZDs have been shown to inhibit proliferation (27), decrease the intimal and medial thickness of carotid arteries in humans (35), and inhibit the development of atherosclerosis (8). In the present study, we found that L-PGDS inhibited PDGF-induced VSMC migration in WKY VSMCs but failed to do so in diabetic GK cells (Fig. 5). This may prove to be detrimental because migration of VSMCs would exacerbate the progression of atherosclerosis. Understanding the mechanisms involved in VSMC migration and ultimately the development of strategies by which this process can be inhibited has been a major focus of research (53). Investigations involving L-PGDS knockouts should prove helpful in defining a more defined role for this protein in atherosclerosis.

Hyperglycemia induces L-PGDS expression in GK VSMCs. Hyperglycemia is a common phenomenon observed in type II diabetes. In this study, we observed a twofold increase in L-PGDS expression in VSMCs isolated from GK diabetic rats cultured under hyperglycemic conditions (Fig. 6). Interestingly, Hirawa et al. (19) found that urinary L-PGDS increased during the early stages of diabetes. When diabetic patient blood sugar levels were controlled, Hamano et al. (18) found that there was a reversal of the elevated urinary L-PGDS levels. In addition, it has been shown that control VSMCs cultured under hyperglycemic conditions will increase cell proliferation (14, 16) and inhibit apoptosis through a PKC-dependent pathway (17). These data correlate well with our previous findings that demonstrated that phorbol ester-induced apoptosis was mediated by L-PGDS phosphorylation and activation by PKC (42) and also our current observation that under hyperglycemic conditions there is a decrease in L-PGDS levels observed in WKY control cells (Fig. 6).

In this study, we have investigated the role of L-PGDS in cell cycle progression, apoptosis, and VSMC migration. We propose that L-PGDS retards cell cycle progression and inhibits the migration of wild-type VSMCs, accentuating the importance of this protein in the cardiovascular complications observed in diabetes. Figure 9 is a proposed mechanism for L-PGDS action. GK diabetic cells seem resistant to the cell cycle inhibition but susceptible to the stimulation of apoptosis via increased L-PGDS uptake. Although this study is limited to tissue culture and the focus is solely on one aspect of atherosclerotic progression, we believe that L-PGDS helps regulate VSMC homeostasis and represents a potential therapeutic target in the treatment of the atherosclerosis associated with diabetes.



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Fig. 9. Role of L-PGDS in atherosclerosis. A proposed mechanism for L-PGDS in the atherosclerotic process is given. PGH2 derived from arachidonic acid is a key intermediate analogous to diacylglycerol (phospholipid metabolism) or glucose 6-phosphate (glucose metabolism). In the presence of L-PGDS, the pool of PGH2 is tilted toward the synthesis of PGD2 and eventually 15-deoxy-{Delta}12,14-prostaglandin J2 (15-dPGJ2), the natural ligand for peroxisome proliferator-activated receptor-{gamma}. 15-dPGJ2 then can arrest VSMC cell growth, stimulate VSMC apoptosis, inhibit VSMC migration, and stimulate macrophage (M{Phi}) differentiation, all of which inhibit atherosclerosis. Mo, monocyte.

 

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 ABSTRACT
 EXPERIMENTAL PROCEDURES
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This laboratory is supported by an American Diabetes Association Career Development Award, American Heart Association Grant-in-Aid 0151192T, National Institutes of Health Grant R01-HL-67953-01A2, and the Winthrop-University Hospital Department of Medicine.


    FOOTNOTES
 

Address for reprint requests and other correspondence: L. Ragolia, Winthrop-Univ. Hospital, Vascular Biology Institute, 222 Station Plaza North, Suite 505-B, Mineola, NY 11501 (E-mail: lragolia{at}winthrop.org)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Balkau B and Eschwege E. Insulin resistance: an independent risk factor for cardiovascular disease? Diabetes Obes Metab 1, Suppl 1: S23–S31, 1999.[ISI][Medline]

2. Begum N, Duddy N, Sandu O, Reinzie J, and Ragolia L. Regulation of myosin-bound protein phosphatase by insulin in vascular smooth muscle cells: evaluation of the role of Rho kinase and phosphatidylinositol-3-kinase-dependent signaling pathways. Mol Endocrinol 14: 1365–1376, 2000.[Abstract/Free Full Text]

3. Begum N and Ragolia L. High glucose and insulin inhibit VSMC MKP-1 expression by blocking iNOS via p38 MAPK activation. Am J Physiol Cell Physiol 278: C81–C91, 2000.[Abstract/Free Full Text]

4. Begum N, Ragolia L, Rienzie J, McCarthy M, and Duddy N. Regulation of mitogen-activated protein kinase phosphatase-1 induction by insulin in vascular smooth muscle cells. Evaluation of the role of the nitric oxide signaling pathway and potential defects in hypertension. J Biol Chem 273: 25164–25170, 1998.[Abstract/Free Full Text]

5. Begum N, Song Y, Rienzie J, and Ragolia L. Vascular smooth muscle cell growth and insulin regulation of mitogen-activated protein kinase in hypertension. Am J Physiol Cell Physiol 275: C42–C49, 1998.[Abstract/Free Full Text]

6. Braun M and Schror K. Prostaglandin D2 relaxes bovine coronary arteries by endothelium-dependent nitric oxide-mediated cGMP formation. Circ Res 71: 1305–1313, 1992.[Abstract]

7. Chen S and Gardner DG. Retinoic acid uses divergent mechanisms to activate or suppress mitogenesis in rat aortic smooth muscle cells. J Clin Invest 102: 653–662, 1998.[Abstract/Free Full Text]

8. Chen Z, Ishibashi S, Perrey S, Osuga J, Gotoda T, Kitamine T, Tamura Y, Okazaki H, Yahagi N, Iizuka Y, Shionoiri F, Ohashi K, Harada K, Shimano H, Nagai R, and Yamada N. Troglitazone inhibits atherosclerosis in apolipoprotein E-knockout mice: pleiotropic effects on CD36 expression and HDL. Arterioscler Thromb Vasc Biol 21: 372–377, 2001.[Abstract/Free Full Text]

9. Cheng ZJ, Vaskonen T, Tikkanen I, Nurminen K, Ruskoaho H, Vapaatalo H, Muller D, Park JK, Luft FC, and Mervaala EM. Endothelial dysfunction and salt-sensitive hypertension in spontaneously diabetic Goto-Kakizaki rats. Hypertension 37: 433–439, 2001.[Abstract/Free Full Text]

10. Collins AR, Meehan WP, Kintscher U, Jackson S, Wakino S, Noh G, Palinski W, Hsueh WA, and Law RE. Troglitazone inhibits formation of early atherosclerotic lesions in diabetic and nondiabetic low density lipoprotein receptor-deficient mice. Arterioscler Thromb Vasc Biol 21: 365–371, 2001.[Abstract/Free Full Text]

11. Craddock BL, Orchiston EA, Hinton HJ, and Welham MJ. Dissociation of apoptosis from proliferation, protein kinase B activation, and BAD phosphorylation in interleukin-3-mediated phosphoinositide 3-kinase signaling. J Biol Chem 274: 10633–10640, 1999.[Abstract/Free Full Text]

12. Diez-Juan A and Andres V. Coordinate control of proliferation and migration by the p27Kip1/cyclin-dependent kinase/retinoblastoma pathway in vascular smooth muscle cells and fibroblasts. Circ Res 92: 402–410, 2003.[Abstract/Free Full Text]

13. Eguchi Y, Eguchi N, Oda H, Seiki K, Kijima Y, Matsu-ura Y, Urade Y, and Hayaishi O. Expression of lipocalin-type prostaglandin D synthase (beta-trace) in human heart and its accumulation in the coronary circulation of angina patients. Proc Natl Acad Sci USA 94: 14689–14694, 1997.[Abstract/Free Full Text]

14. Fujita N, Furukawa Y, Du J, Itabashi N, Fujisawa G, Okada K, Saito T, and Ishibashi S. Hyperglycemia enhances VSMC proliferation with NF-{kappa}B activation by angiotensin II and E2F-1 augmentation by growth factors. Mol Cell Endocrinol 192: 75–84, 2002.[CrossRef][ISI][Medline]

15. Goto Y, Kakizaki M, and Masaki N. Production of spontaneous diabetic rats by repetition of selective breeding. Tohoku J Exp Med 119: 85–90, 1976.[ISI][Medline]

16. Hall JL, Chatham JC, Eldar-Finkelman H, and Gibbons GH. Upregulation of glucose metabolism during intimal lesion formation is coupled to the inhibition of vascular smooth muscle cell apoptosis. Role of GSK3{beta}. Diabetes 50: 1171–1179, 2001.[Abstract/Free Full Text]

17. Hall JL, Matter CM, Wang X, and Gibbons GH. Hyperglycemia inhibits vascular smooth muscle cell apoptosis through a protein kinase C-dependent pathway. Circ Res 87: 574–580, 2000.[Abstract/Free Full Text]

18. Hamano K, Totsuka Y, Ajima M, Gomi T, Ikeda T, Hirawa N, Eguchi Y, Yamakado M, Takagi M, Nakajima H, Oda H, Seiki K, Eguchi N, Urade Y, and Uehara Y. Blood sugar control reverses the increase in urinary excretion of prostaglandin D synthase in diabetic patients. Nephron 92: 77–85, 2002.[CrossRef][ISI][Medline]

19. Hirawa N, Uehara Y, Ikeda T, Gomi T, Hamano K, Totsuka Y, Yamakado M, Takagi M, Eguchi N, Oda H, Seiki K, Nakajima H, and Urade Y. Urinary prostaglandin D synthase ({beta}-trace) excretion increases in the early stage of diabetes mellitus. Nephron 87: 321–327, 2001.[CrossRef][ISI][Medline]

20. Hirawa N, Uehara Y, Yamakado M, Toya Y, Gomi T, Ikeda T, Eguchi Y, Takagi M, Oda H, Seiki K, Urade Y, and Umemura S. Lipocalin-type prostaglandin D synthase in essential hypertension. Hypertension 39: 449–454, 2002.[Abstract/Free Full Text]

21. Hsueh WA and Law RE. PPAR{gamma} and atherosclerosis: effects on cell growth and movement. Arterioscler Thromb Vasc Biol 21: 1891–1895, 2001.[Abstract/Free Full Text]

22. Hupfeld CJ and Weiss RH. TZDs inhibit vascular smooth muscle cell growth independently of the cyclin kinase inhibitors p21 and p27. Am J Physiol Endocrinol Metab 281: E207–E216, 2001.[Abstract/Free Full Text]

23. Inoue T, Takayanagi K, Morooka S, Uehara Y, Oda H, Seiki K, Nakajima H, and Urade Y. Serum prostaglandin D synthase level after coronary angioplasty may predict occurrence of restenosis. Thromb Haemost 85: 165–170, 2001.[ISI][Medline]

24. Janssen U, Riley SG, Vassiliadou A, Floege J, and Phillips AO. Hypertension superimposed on type II diabetes in Goto Kakizaki rats induces progressive nephropathy. Kidney Int 63: 2162–2170, 2003.[CrossRef][ISI][Medline]

25. Jung F, Haendeler J, Goebel C, Zeiher AM, and Dimmeler S. Growth factor-induced phosphoinositide 3-OH kinase/Akt phosphorylation in smooth muscle cells: induction of cell proliferation and inhibition of cell death. Cardiovasc Res 48: 148–157, 2000.[CrossRef][ISI][Medline]

26. Law RE, Goetze S, Xi XP, Jackson S, Kawano Y, Demer L, Fishbein MC, Meehan WP, and Hsueh WA. Expression and function of PPAR{gamma} in rat and human vascular smooth muscle cells. Circulation 101: 1311–1318, 2000.[Abstract/Free Full Text]

27. Law RE, Meehan WP, Xi XP, Graf K, Wuthrich DA, Coats W, Faxon D, and Hsueh WA. Troglitazone inhibits vascular smooth muscle cell growth and intimal hyperplasia. J Clin Invest 98: 1897–1905, 1996.[Abstract/Free Full Text]

28. Liakopoulos D, Busgen T, Brychzy A, Jentsch S, and Pause A. Conjugation of the ubiquitin-like protein NEDD8 to cullin-2 is linked to von Hippel-Lindau tumor suppressor function. Proc Natl Acad Sci USA 96: 5510–5515, 1999.[Abstract/Free Full Text]

29. Lundberg MS, Curto KA, Bilato C, Monticone RE, and Crow MT. Regulation of vascular smooth muscle migration by mitogen-activated protein kinase and calcium/calmodulin-dependent protein kinase II signaling pathways. J Mol Cell Cardiol 30: 2377–2389, 1998.[CrossRef][ISI][Medline]

30. Maesaka JK, Palaia T, Frese L, Fishbane S, and Ragolia L. Prostaglandin D(2) synthase induces apoptosis in pig kidney LLC-PK1 cells. Kidney Int 60: 1692–1698, 2001.[CrossRef][ISI][Medline]

31. Marks JB and Raskin P. Cardiovascular risk in diabetes: a brief review. J Diabetes Complications 14: 108–115, 2000.[CrossRef][ISI][Medline]

32. Marx N, Schonbeck U, Lazar MA, Libby P, and Plutzky J. Peroxisome proliferator-activated receptor gamma activators inhibit gene expression and migration in human vascular smooth muscle cells. Circ Res 83: 1097–1103, 1998.[Abstract/Free Full Text]

33. Matsuoka T, Hirata M, Tanaka H, Takahashi Y, Murata T, Kabashima K, Sugimoto Y, Kobayashi T, Ushikubi F, Aze Y, Eguchi N, Urade Y, Yoshida N, Kimura K, Mizoguchi A, Honda Y, Nagai H, and Narumiya S. Prostaglandin D2 as a mediator of allergic asthma. Science 287: 2013–2017, 2000.[Abstract/Free Full Text]

34. Minami T, Okuda-Ashitaka E, Nishizawa M, Mori H, and Ito S. Inhibition of nociceptin-induced allodynia in conscious mice by prostaglandin D2. Br J Pharmacol 122: 605–610, 1997.[Abstract]

35. Minamikawa J, Yamauchi M, Inoue D, and Koshiyama H. Another potential use of troglitazone in noninsulin-dependent diabetes mellitus. J Clin Endocrinol Metab 83: 1041–1042, 1998.[Free Full Text]

36. Miwa Y, Sasaguri T, Inoue H, Taba Y, Ishida A, and Abumiya T. 15-Deoxy-{Delta}12,14-prostaglandin J(2) induces G(1) arrest and differentiation marker expression in vascular smooth muscle cells. Mol Pharmacol 58: 837–844, 2000.[Abstract/Free Full Text]

37. Ohh M, Kim WY, Moslehi JJ, Chen Y, Chau V, Read MA, and Kaelin WG Jr. An intact NEDD8 pathway is required for Cullin-dependent ubiquitylation in mammalian cells. EMBO Rep 3: 177–182, 2002.[Abstract/Free Full Text]

38. Pan ZQ, Kentsis A, Dias DC, Yamoah K, and Wu K. Nedd8 on cullin: building an expressway to protein destruction. Oncogene 23: 1985–1997, 2004.[CrossRef][ISI][Medline]

39. Patel VA, Zhang QJ, Siddle K, Soos MA, Goddard M, Weissberg PL, and Bennett MR. Defect in insulin-like growth factor-1 survival mechanism in atherosclerotic plaque-derived vascular smooth muscle cells is mediated by reduced surface binding and signaling. Circ Res 88: 895–902, 2001.[Abstract/Free Full Text]

40. Perlman H, Sata M, Le Roux A, Sedlak TW, Branellec D, and Walsh K. Bax-mediated cell death by the Gax homeoprotein requires mitogen activation but is independent of cell cycle activity. EMBO J 17: 3576–3586, 1998.[Abstract/Free Full Text]

41. Ragolia L, Palaia T, Frese L, Fishbane S, and Maesaka JK. Prostaglandin D2 synthase induces apoptosis in PC12 neuronal cells. Neuroreport 12: 2623–2628, 2001.[CrossRef][ISI][Medline]

42. Ragolia L, Palaia T, Paric E, and Maesaka JK. Elevated L-PGDS activity contributes to PMA-induced apoptosis concomitant with downregulation of PI3-K. Am J Physiol Cell Physiol 284: C119–C126, 2003.[Abstract/Free Full Text]

43. Ragolia L, Palaia T, Paric E, and Maesaka JK. Prostaglandin D2 synthase inhibits the exaggerated growth phenotype of spontaneously hypertensive rat vascular smooth muscle cells. J Biol Chem 278: 22175–22181, 2003.[Abstract/Free Full Text]

44. Rogers MS, Rohan RM, Birsner AE, and D'Amato RJ. Genetic loci that control vascular endothelial growth factor-induced angiogenesis. FASEB J 17: 2112–2114, 2003.[Abstract/Free Full Text]

45. Sandu OA, Ragolia L, and Begum N. Diabetes in the Goto-Kakizaki rat is accompanied by impaired insulin-mediated myosin-bound phosphatase activation and vascular smooth muscle cell relaxation. Diabetes 49: 2178–2189, 2000.[Abstract]

46. Sedding DG, Seay U, Fink L, Heil M, Kummer W, Tillmanns H, and Braun-Dullaeus RC. Mechanosensitive p27Kip1 regulation and cell cycle entry in vascular smooth muscle cells. Circulation 108: 616–622, 2003.[Abstract/Free Full Text]

47. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, and Klenk DC. Measurement of protein using bicinchoninic acid. Anal Biochem 150: 76–85, 1985.[ISI][Medline]

48. Suhara T, Kim HS, Kirshenbaum LA, and Walsh K. Suppression of Akt signaling induces Fas ligand expression: involvement of caspase and Jun kinase activation in Akt-mediated Fas ligand regulation. Mol Cell Biol 22: 680–691, 2002.[Abstract/Free Full Text]

49. Taba Y, Sasaguri T, Miyagi M, Abumiya T, Miwa Y, Ikeda T, and Mitsumata M. Fluid shear stress induces lipocalin-type prostaglandin D(2) synthase expression in vascular endothelial cells. Circ Res 86: 967–973, 2000.[Abstract/Free Full Text]

50. Tanaka T, Urade Y, Kimura H, Eguchi N, Nishikawa A, and Hayaishi O. Lipocalin-type prostaglandin D synthase (beta-trace) is a newly recognized type of retinoid transporter. J Biol Chem 272: 15789–15795, 1997.[Abstract/Free Full Text]

51. Urade Y and Hayaishi O. Prostaglandin D synthase: structure and function. Vitam Horm 58: 89–120, 2000.[CrossRef][ISI][Medline]

51. Wakino S, Kintscher U, Liu Z, Kim S, Yin F, Ohba M, Kuroki T, Schonthal AH, Hsueh WA, and Law RE. Peroxisome proliferator-activated receptor gamma ligands inhibit mitogenic induction of p21(Cip1) by modulating the protein kinase Cdelta pathway in vascular smooth muscle cells. J Biol Chem 276: 47650–47657, 2001.[Abstract/Free Full Text]

52. Whittle BJ, Moncada S, and Vane JR. Comparison of the effects of prostacyclin (PGI2), prostaglandin E1 and D2 on platelet aggregation in different species. Prostaglandins 16: 373–388, 1978.[CrossRef][Medline]

53. Willis AIPPD, Sumpio BE, Gahtan V. Vascular smooth muscle cell migration: current research and clinical implications. Vasc Endovascular Surg 38: 11–23, 2004.[Medline]

54. Yano K, Bauchat JR, Liimatta MB, Clemmons DR, and Duan C. Down-regulation of protein kinase C inhibits insulin-like growth factor I-induced vascular smooth muscle cell proliferation, migration, and gene expression. Endocrinology 140: 4622–4632, 1999.[Abstract/Free Full Text]

55. Zhang X, Zhang Q, Li P, Yu B, Wang T, Zhu Y, Zhang Y, and Cai D. [Inhibition of proliferation of human vascular smooth muscle cells by overexpression of p21 gene]. Zhonghua Yi Xue Za Zhi 82: 696–698, 2002.[Medline]





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