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Am J Physiol Heart Circ Physiol 290: H1337-H1346, 2006. First published January 6, 2006; doi:10.1152/ajpheart.00936.2005
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Regulation of Cardiovascular Functions by Eicosanoids and Other Lipid Mediators

The prostacyclin receptor induces human vascular smooth muscle cell differentiation via the protein kinase A pathway

Kristina M. Fetalvero,1,2 Maureen Shyu,2 Athena P. Nomikos,1 Yuh-Fang Chiu,2 Robert J. Wagner,2 Richard J. Powell,2 John Hwa,1,3 and Kathleen A. Martin1,2

1Department of Pharmacology and Toxicology, Dartmouth Medical School; and 2Department of Surgery, Section of Vascular Surgery, Dartmouth-Hitchcock Medical Center; and 3Department of Medicine, Section of Cardiology, Dartmouth Medical School, Lebanon, New Hampshire

Submitted 1 September 2005 ; accepted in final form 14 November 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Recent studies of cyclooxygenase-2 (COX-2) inhibitors suggest that the balance between thromboxane and prostacyclin is a critical factor in cardiovascular homeostasis. Disruption of prostacyclin signaling by genetic deletion of the receptor or by pharmacological inhibition of COX-2 is associated with increased atherosclerosis and restenosis after injury in animal models and adverse cardiovascular events in clinical trials (Vioxx). Human vascular smooth muscle cells (VSMC) in culture exhibit a dedifferentiated, migratory, proliferative phenotype, similar to what occurs after arterial injury. We report that the prostacyclin analog iloprost induces differentiation of VSMC from this synthetic, proliferative phenotype to a quiescent, contractile phenotype. Iloprost induced expression of smooth muscle (SM)-specific differentiation markers, including SM-myosin heavy chain, calponin, h-caldesmon, and SM {alpha}-actin, as determined by Western blotting and RT-PCR analysis. Iloprost activated cAMP/protein kinase A (PKA) signaling in human VSMC, and the cell-permeable cAMP analog 8-bromo-cAMP mimicked the iloprost-induced differentiation. Both myristoylated PKA inhibitor amide-(14–22) (PKI, specific PKA inhibitor), as well as ablation of the catalytic subunits of PKA by small interfering RNA, opposed the upregulation of contractile markers induced by iloprost. These data suggest that iloprost modulates VSMC phenotype via Gs activation of the cAMP/PKA pathway. These studies reveal regulation of VSMC differentiation as a potential mechanism for the cardiovascular protective effects of prostacyclin. This provides important mechanistic insights into the induction of cardiovascular events with the use of selective COX-2 inhibitors.

iloprost; smooth muscle-specific differentiation markers; phenotypic modulation; signal transduction; intimal hyperplasia

PHENOTYPIC MODULATION of vascular smooth muscle cells (VSMC) is critical in a number of normal and pathophysiological processes, including angiogenesis, atherosclerosis, and restenosis. VSMC retain the plasticity to dedifferentiate to a synthetic phenotype in response to growth factor stimulation or vascular injury (25). In a normal, mature blood vessel, VSMC exhibit a "contractile" differentiated phenotype, characterized by expression of smooth muscle (SM)-specific contractile proteins, such as SM-myosin heavy chain (MHC), and calponin. This allows contractile function to regulate blood vessel tone, blood pressure, and flow (23). Dedifferentiated VSMC downregulate expression of SM-specific contractile proteins and gain the ability to proliferate, migrate, and upregulate extracellular matrix (ECM) protein synthesis and are thus referred to as "synthetic" phenotype (25, 28). Endothelial dysfunction associated with atherosclerosis leads to a loss of endothelium-derived factors such as prostacyclin (PGI2) and nitric oxide (1, 8, 11, 19, 33). Loss of such factors, as well as increased exposure to growth factors, contributes to VSMC dedifferentiation from contractile to synthetic phenotype, leading to intimal thickening of the affected artery. The hormonal factors and mechanisms underlying VSMC phenotypic modulation are currently being intensively explored. An understanding of these fundamental processes may provide therapeutic targets to prevent vascular disease and its progression.

PGI2, a 20-carbon prostanoid derivative of arachidonic acid metabolism, is the main cyclooxygenase-2 (COX-2) product in vascular endothelial cells (17, 32). PGI2 mediates its cellular activity via the tissue-specific, cell surface G protein-coupled receptor, the prostacyclin receptor IP (International Union of Pharmacology Receptor classification) (2). IP, expressed on both VSMC and platelets, predominantly couples to the heterotrimeric G protein Gs, leading to activation of adenylyl cyclase (AC), formation of the second messenger cAMP, and activation of the cAMP-dependent protein kinase, PKA (13, 30). Because prostacyclin is a vasodilator as well as a potent inhibitor of platelet activation and of VSMC proliferation, it is considered an important endogenous factor that protects blood vessels from cardiovascular disease. Reduced PGI2 activity has been implicated in various cardiovascular disorders, such as atherosclerosis, myocardial infarction, thrombosis, and myocardial ischemia and pulmonary hypertension, and the PGI2 analog iloprost is used clinically to treat pulmonary hypertension (21). Notably, genetic deletion of the prostacyclin receptor in mice is associated with increased injury-induced restenosis, thrombotic events, and atherosclerosis (5, 7, 20).

Recent studies of selective COX-2 inhibitors suggest that the balance between prostacyclin and thromboxane (TxA2) is a critical factor in cardiovascular homeostasis (34). TxA2 is a derivative of COX-1-catalyzed arachidonic acid metabolism released by activated platelets (10). Unlike PGI2, TxA2 is a potent activator of platelets and a vasoconstrictor (27). Genetic deletion of both the IP and the TxA2 receptor (TP) in mice provided evidence that PGI2 modulates cardiovascular homeostasis in vivo by regulating the response to TxA2 (5). Additionally, studies have shown that selective inhibition of COX-2 leads to a decrease in PGI2 production with no effect on TxA2 production (17). More recently, clinical trials of COX-2 inhibitors, such as rofecoxib and celecoxib, suggest that pharmacological inhibition of COX-2 is associated with adverse cardiovascular events, possibly through a disruption of the homeostatic balance between PGI2 and TxA2 (34).

The goals of this study were to determine the role of prostacyclin receptor signaling in modulating human VSMC differentiation and to define the mechanism involved in this process. We report that activation of the human prostacyclin receptor with the stable prostacyclin mimetic iloprost induces a differentiated phenotype in human VSMC through a mechanism that is dependent on production of cAMP and subsequent activation of PKA. Regulation of VSMC phenotype may be another potential mechanism for the cardiovascular protective effects of prostacyclin.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. Human VSMC were isolated by explant method from iliac or aortic arterial samples from organ donors or superficial femoral arteries obtained after lower extremity amputation for peripheral vascular occlusive disease (9), in accordance with Institutional Review Board-approved protocols. The cells were cultured in medium 199 (M199) supplemented with 10% fetal bovine serum (FBS), L-glutamine, penicillin-streptomycin, fibroblast growth factor (Promega, Madison, WI), and epidermal growth factor. Bovine aortic VSMC prepared by explant were cultured in DMEM with 10% FBS. Passages 3–7 were used for experiments. All drug treatments were performed in M199 containing 2.5% FBS. Under these serum conditions, VSMC continue to proliferate at a reduced rate and do not spontaneously differentiate (3). Vehicle-treated cells were incubated with PBS for the maximum duration of the experimental treatment. VSMC were treated with various drugs as indicated in all the figure legends. Iloprost was purchased from Amersham Biosciences (Pittsburgh, PA). 8-Bromo-cAMP (8-Br-cAMP) and carbacyclin were purchased from Sigma-Aldrich (St. Louis, MO). Cicaprost was a kind gift from Shering AG (Berlin, Germany). Rapamycin and the cell-permeable myristoylated PKA inhibitor-(14–22) amide (myrPKI) were purchased from Calbiochem (San Diego, CA).

Determination of cAMP levels. At confluence, VSMC in 12-well plates were incubated with M199 containing 2.5% FBS for 18 h. Cells were washed twice with PBS plus 4 mmol/l EDTA and 2 mmol/l IBMX (pH 7.4) and incubated at 20°C for 10 min. Defined concentrations of prostacyclin analogs iloprost, cicaprost, or carbacyclin (10 pmol/l to 10 µmol/l) were added to the cells for 20 min. For the time-course experiment, cells were instead treated with 2.5 nmol/l iloprost for the times indicated (1 min–24 h). Cells were harvested and boiled for 3 min, followed by high-speed (10,000 rpm) centrifugation. cAMP production was determined using a radioreceptor competition assay (Amersham Biosciences). In brief, [3H]cAMP was used in competition for a cAMP-binding protein against known concentrations of nonradiolabeled cAMP, followed by determination of the unknowns. The reaction proceeded for 2 h at 4°C. Excess unbound cAMP was removed with charcoal. Samples were counted in 5 ml of Liquiscint (National Diagnostics). Results were analyzed using GraphPad Prism software. For the concentration response, a nonlinear, curve-fitting program (GraphPad Prism) was used, and the EC50 was determined. ANOVA (posttest Newman-Keuls) and Student's t-tests were used to determine statistically significant differences (P < 0.05).

Semiquantitative RT-PCR. After treatment with appropriate agonists, total RNA was isolated using the Qiagen RNeasy kit with DNase I (Qiagen, Valencia, CA) and quantitated in duplicate by spectrophotometry. RNA (0.5–1 µg) was reverse transcribed using MMLV RNase H+ reverse transcriptase and a blend of oligo(dT) and random hexamer primers (iScript, Bio-Rad, Hercules, CA). Primers were designed to specifically amplify the human basic calponin gene transcript (sense 5'-TAACCGAGGTCCTGCCTACG, antisense 5'-TGTGGGTGGGCTCACTCAGC), the SM-MHC transcript (sense 5'-CGCTGAATGACAACGTGACTTCC, antisense 5'-CCAGTTCCGCAGCTTGAGGTA), and the pyruvate dehydrogenase (PDH) transcript (housekeeping control gene) (sense 5'-AGCTGCCAAGACCTACTACAT, antisense 5'-ATCCCGAATGGCTGATTT). To determine the linear range of the PCR for each primer set, titrations using dilutions of the reverse transcribed cDNA were performed. PCR was performed using 20 pmol of each primer, 0.04 mmol/l dNTPs, Red Taq DNA polymerase (Sigma), and TaqStart antibody (Clontech, Mountain View, CA) or HotMaster Taq DNA polymerase (Eppendorf, Westbury, NY). PCR with calponin and PDH primer sets were performed for 30 cycles: 95°C (30 s), 58.5°C (calponin) or 52.4°C (PDH) (30 s), and 70°C (1 min). PCR with MHC primers was performed for 32 cycles: 95°C (30 s), 57.6°C (30 s), and 70°C (1 min). PCR products were resolved on 1% agarose gels with ethidium bromide (Sigma) or GelStar nucleic acid stain (Cambrex). A digital image was obtained using a Typhoon Scanner (Molecular Dynamics), and relative quantities of product were determined using Scion Image Beta 4.0.2. Contractile protein product was normalized to PDH. Results were analyzed using GraphPad Prism software. An ANOVA (multiple comparison post hoc test Newman-Keuls) was used to determine statistically significant differences (P < 0.05).

Cell lysis and immunoblotting. Cells were washed with PBS and scraped in lysis buffer [10 mmol/l K3PO4, 1 mmol/l EDTA, 10 mmol/l MgCl2, 50 mmol/l beta-glycerophosphate, 5 mmol/l EGTA, 0.5% (vol/vol) Nonidet P-40, 0.1% Brij 35, 0.1% sodium deoxycholate, 1 mmol/l sodium orthovanadate, complete protease inhibitors tablet (Roche, Indianapolis, IN)]. Extracts were subjected to one freeze/thaw cycle at –80°C and centrifuged at 16,000 g for 30 s. Total protein concentration in each lysate (supernatant) was determined by Bradford assay (Bio-Rad). Equal amounts of protein (8–20 µg) from each lysate were loaded per lane and separated by 7.5% or 12% SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted using primary antibodies against SM {alpha}-actin, calponin, h-caldesmon (Sigma), smooth muscle MHC isoform SM2 (SM2-MHC) (Seikagaku America), or GAPDH (AbCam, Cambridge, MA). After immunoblotting with horseradish peroxidase-conjugated secondary antibody, signal intensity was detected with enhanced chemiluminescence reagents (Pierce, Rockford, IL). Relative quantities of protein were determined using Scion Image Beta 4.0.2 and normalized to GAPDH. Results were analyzed using GraphPad Prism software. ANOVA (multiple comparison post hoc test Newman-Keuls) was used to determine statistically significant differences (P < 0.05).

Cell area measurement. VSMC were plated on coverslips at a density of 104–105 cells per coverslip. After 18 h, the cells were grown in M199 containing 2.5% FBS with vehicle or appropriate agonist for 48 h. Cells were fixed with methanol:acetone (1:1), stained with toluidine blue, and mounted on slides. Four separate high-power fields from each slide were photographed, and cell area was determined in a blinded manner by planimetry using NIH Image software to outline cell dimensions and compute two-dimensional cell area. Rapamycin treatment served as a positive control (16). A Student's t-test (GraphPad Prism) was used to determine statistical significance (P < 0.05).

Nonradioactive in vitro assay for PKA activity. VSMC were stimulated as indicated in Figs. 5–7. Cells were lysed as described for Western blotting, and total protein concentration in each lysate was determined by Bradford assay. Equal amounts of protein from each sample (25–30 µg) were subject to the PepTag assay for nonradioactive detection of cAMP-dependent protein kinase (Promega), according to the manufacturer's protocol. In brief, total cell lysates were subjected to a kinase reaction with 2 µg of the fluorescence-labeled peptide substrate kemptide for 30 min. For analysis of Michaelis-Menten kinetics, substrate concentrations were varied from 0.25 to 6 µg. The reaction was terminated by boiling the samples for 10 min. The phosphorylated and nonphosphorylated kemptides were separated on a 0.8% agarose gel, and an image was obtained using a Typhoon Scanner (Molecular Dynamics). Quantities of phosphorylated substrate were determined using Scion Image Beta 4.0.2. Results were analyzed using GraphPad Prism software. An ANOVA (multiple comparison post hoc test Newman-Keuls) was used to determine statistically significant differences (P < 0.05).


Figure 5
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  		Fig. 5. Iloprost induces PKA activity in human VSMC. A: cell lysates were prepared from human VSMC treated with the indicated concentrations of iloprost or vehicle for 20 min and analyzed for PKA activity using a nonradioactive in vitro PKA assay. Bar graph shows densitometric quantitation of fluorescence units of phosphorylated kemptide (P-kemptide) from 3 independent experiments expressed as fold induction relative to control. Results are expressed as means ± SE, and P values for Newman-Keuls post hoc test are indicated above the bars: *P < 0.05 and ***P < 0.001 relative to vehicle control. A representative experiment is shown. EC50 was determined from best-fit curve with nonlinear regression. Kemptide, PKA substrate with fluorescent label. B: kinase assay was performed (in duplicate) as above using a constant 27 µg of lysate per reaction and increasing concentrations of kemptide substrate. PKA activity is plotted relative to the substrate concentration. Inset: Lineweaver-Burke plot (data are means ± SE). C: kinase assay was performed as above using 2 µg kemptide and increasing amounts of cell lysate (5–40 µg).

 

Figure 7
Figure 7
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  		Fig. 7. Inhibition of PKA opposes iloprost-induced VSMC differentiation. A: human VSMC were preincubated with 5 µmol/l myristoylated PKA inhibitor (myrPKI) for 30 min followed by treatment with 2.5 nmol/l iloprost for 45 min. Cell lysates were analyzed for PKA activity using a nonradioactive in vitro PKA assay. A representative experiment is shown; veh, vehicle. B–D: Western blot analysis of cell lysates from human VSMC pretreated for 30 min with 5 µmol/l myrPKI with or without 2.5 nmol/l iloprost for 18 h. Antibodies against SM-specific differentiation markers SM2-MHC (B), h-caldesmon (C), and calponin (D) were used, and representative blots are shown. Bar graph shows densitometric quantitation as means ± SE of 3 independent experiments in which each treatment group was performed in triplicate. P values were generated by one-way ANOVA with Newman-Keuls multiple comparison post hoc test and are indicated above the bars: **P < 0.01, ***P < 0.001 relative to vehicle (veh) and {dagger}{dagger}P < 0.01, {dagger}{dagger}{dagger}P < 0.001 relative to iloprost. E: human VSMC were transiently transfected with the indicated concentrations of a nonsilencing siCONTROL or small interfering RNA (siRNA) targeted against PKA catalytic subunits-{alpha} and -beta (PKA-C{alpha} + PKA-Cbeta). Cell lysates were harvested 40 h posttransfection and were subjected to an in vitro PKA assay. F: VSMC were transiently transfected with siCONTROL or siRNA to PKA-C{alpha} and -Cbeta as above (0.25 µg each). At 24 h posttransfection, cells were treated with 2.5 nmol/l iloprost for an additional 6 h. Total RNA was isolated and subjected to RT-PCR by using primers to PKA-C{alpha} and -Cbeta to confirm gene knockdown. The samples were also subjected to RT-PCR by using primers to the calponin, SM-MHC (MHC), and PDH genes. PDH was used as a loading control. Representative data from 2 independent experiments are shown.

 
Transient transfection of small interfering RNA. Human VSMC (8 x 105 cells) were transfected with 40 pmol small interfering RNA (siRNA) using the basic smooth muscle cell (SMC) Nucleofector solution and the Nucleofector device set at program A-33 (Amaxa, Gaithersburg, MD). After transfection, the cells were treated with vehicle or iloprost for 6 h and harvested for RT-PCR analysis and PKA kinase assays.

The sequence for PKA catalytic subunit-{alpha} (PKA-C{alpha}) siRNA was 5'-AAGUGGUUUGCCACAACUGAC-3' and the siRNA sequence for PKA catalytic subunit-beta (PKA-Cbeta) was 5'-AAGAGUUUCUAGCCAAAGCCA-3' (Dharmacon, Lafayette, CO). Nontargeting duplex siRNA (siCONTROL) (Dharmacon) was used as a negative control. Human VSMC were transfected with 20–40 pmol (0.25–0.5 µg) of PKA-C{alpha} and 20–40 pmol PKA-Cbeta siRNA or 40–80 pmol (0.5–1.0 µg) of the siCONTROL. To confirm gene knockdown, total RNA was isolated from cells transiently transfected with PKA-specific or nontargeting siRNA and subjected to RT-PCR (as described above) using primers specific for PKA-C{alpha} transcript (sense 5'-CTTTCCTCCCAGCAGCGTTTC, antisense 5'-TTCAAG AGGAAGTCTCGTCC) or primers for PKA-Cbeta transcript (sense 5'-CCAAGAACATGCTGACCAATG, antisense 5'-TTCTTGGCTAAATTGTGGCTGA). PDH was assayed as a control.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Iloprost induces cAMP signaling in human VSMC. It is well-established that stimulation of the prostacyclin receptor by the endogenous ligand prostacyclin and its analogs results in the receptor coupling to the G protein Gs, which leads to an accumulation of the second messenger cAMP and ultimately activation of PKA (13). However, because of the technical challenges in obtaining, culturing, and modulating the phenotype of human VSMC, the majority of the in vitro studies of prostacyclin signaling have been performed in VSMC from animal models. Because emerging evidence points to a critical role for prostacyclin signaling in human cardiovascular physiology, experiments were performed in human VSMC. First, the ability of the prostacyclin receptor to couple to Gs in human VSMC stimulated with iloprost was assessed by measuring accumulation of cAMP. Because of the very short PGI2 half-life (3 min), we employed more stable prostacyclin analogs (11). Treating VSMC with increasing concentrations of three prostacyclin analogs (iloprost, cicaprost, and carbacyclin) led to a concentration-dependent increase in cAMP production (Fig. 1A). The maximal amount of cAMP (pmol) was generated in response to 100 nmol/l iloprost with an EC50 of 8 nmol/l. Similar results were obtained with cicaprost, whereas carbacyclin was slightly less potent than either iloprost or cicaprost with an EC50 of 300 nmol/l (Fig. 1A). Iloprost is the most stable prostacyclin analog approved for clinical use. Therefore, iloprost was used for differentiation assays, at concentrations near the EC50 for cAMP production. To determine the duration of prostacyclin receptor signaling, we measured cAMP levels after treating cells from 1 min to 24 h with 2.5 nmol/l iloprost. cAMP was generated in response to iloprost for up to 90 min (Fig. 1B).


Figure 1
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  		Fig. 1. Prostacyclin agonists stimulate cAMP accumulation in human vascular smooth muscle cells (VSMC). A: concentration-response curves were determined by the addition of concentrations of iloprost, cicaprost, or carbacyclin, ranging from 10 µmol/l to 10 pmol/l. After 20 min, cells were harvested, and cAMP was measured using a competition assay as described in MATERIALS AND METHODS. Results are means ± SE (pmol of cAMP). EC50 was determined from best-fit curve with nonlinear regression. B: cells were treated with 2.5 nmol/l iloprost, and the change in cAMP ({Delta}cAMP) was determined at the times indicated on the graph (1 min–24 h) by using the assay described above. Background subtraction was performed using parallel plates (duplicate) not treated with iloprost, in addition to cAMP determined for the previous time point. No further increase in cAMP was observed after 90 min. Data are means ± SE (pmol of cAMP/well).

 
Iloprost induces SM-specific differentiation markers. VSMC in a mature adult vessel are maintained in a differentiated phenotype in which the cells express a repertoire of SM-specific proteins required for normal cardiovascular homeostasis and function (25). On vessel injury, these cells proliferate, migrate into the intima, lose expression of contractile proteins, synthesize extracellular matrix, and lose expression of contractile proteins. Passaged VSMC in culture mimic this synthetic, dedifferentiated state (28).

We tested the hypothesis that iloprost would induce VSMC differentiation by treating synthetic phenotype human VSMC in culture with iloprost and measuring SM-specific contractile protein expression, well-established biochemical markers of differentiation (25, 28). Iloprost treatment (2.5 nmol/l) resulted in a rapid (2 h) sevenfold upregulation of transcript levels of SM-MHC, the most stringent marker of SMC differentiation (15), as measured by semiquantitative RT-PCR (Fig. 2A). The iloprost-induced increase in SM-MHC transcript persisted for 14 h (Fig. 2A). The differentiation marker calponin was similarly upregulated by iloprost, with slightly different kinetics (Fig. 2, B and C). Iloprost induced a twofold upregulation at 2 h that persisted for up to 24 h, with a maximal 10-fold induction after 18 h (Fig. 2B). The upregulation of differentiation marker transcripts was accompanied by an increase in the contractile marker protein expression (Fig. 3, A–F). As shown in Fig. 3, A–C, increasing concentrations of iloprost resulted in an increase in the expression of calponin, SM {alpha}-actin, and SM2-MHC that was near maximal with 2.5 nmol/l iloprost. Furthermore, iloprost induced a time-dependent accumulation of all SM-specific differentiation marker proteins assayed, including calponin, h-caldesmon, and SM2-MHC (Fig. 3, D–F). Notably, the upregulation of calponin and SM2-MHC protein closely followed the kinetics of the increase in the corresponding transcripts.


Figure 2
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  		Fig. 2. Iloprost upregulates the expression of smooth muscle (SM)-specific differentiation marker mRNAs. A: human VSMC were cultured in the presence of 2.5 nmol/l iloprost over the time course indicated. Vehicle control ("0") was harvested 18 h after PBS treatment. Total RNA was prepared and subjected to reverse transcription and semiquantitative PCR (RT-PCR) by using primers to the SM-myosin heavy chain (MHC) gene. The samples were also subjected to RT-PCR using primers to pyruvate dehydrogenase (PDH) as a control. Gels from a representative experiment are shown. Bar graph shows densitometric quantitation as means ± SE of 2 independent experiments, in which each time point was performed in at least duplicate. P values were generated by one-way ANOVA with Newman-Keuls multiple comparison post hoc test and are indicated above the bars: *P < 0.05, **P < 0.01 relative to vehicle. AU, arbitrary units. B: total RNA was isolated from human VSMC treated with 2.5 nmol/l iloprost for the times indicated, and semiquantitative RT-PCR was performed using primers to the calponin or PDH gene. Vehicle control ("0") was harvested 24 h after PBS treatment. A representative experiment is shown. Bar graph shows corresponding densitometric data (in AU) with calponin normalized to PDH. C: semiquantitative RT-PCR for calponin and PDH (as above) for human VSMC treated with 2.5 nmol/l iloprost or vehicle for 6 h. Duplicates are shown in the gels, and means ± SE are shown graphically.

 

Figure 3
Figure 3
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  		Fig. 3. Iloprost upregulates expression of SM-specific contractile differentiation marker protein. A: human VSMC were treated with the indicated concentrations of iloprost or vehicle for 6 h. Cell lysates were prepared, and Western blot analysis was performed using antibodies against SM {alpha}-actin and calponin. B and C: human VSMC were treated with the indicated concentrations of iloprost or vehicle for 18 h and analyzed by Western blotting as above for SM-MHC isotype SM2 (SM2-MHC) (B) or SM {alpha}-actin (C). GAPDH blots are shown as controls for lane loading. D–F: Western blot analysis of contractile proteins was performed from cell lysates prepared from human VSMC treated with 2.5 nmol/l iloprost for the time periods indicated. Vehicle-treated cells were harvested after 24 h. Representative blots for calponin (D), h-caldesmon (E), and SM-MHC isotype SM2 (SM2-MHC) (F) are shown. GAPDH was used as a loading control. The corresponding densitometry data are mean values ± SE from 3 replicate experiments where each time point was performed in triplicate and are represented in the graph expressed as fold induction relative to control. P values were generated using one-way ANOVA with post hoc test Newman-Keuls and are indicated above the bars: *P < 0.05, **P < 0.01, ***P < 0.001 relative to vehicle.

 
Iloprost induces a contractile morphology in VSMC. Passaged VSMC in culture in the synthetic phenotype are characterized by a large, flattened, fibroblast-like morphology, in contrast to the narrow, elongated, "spindle-shaped" morphology of contractile phenotype VSMC in primary culture (12). We have previously shown that this morphological difference can be quantitatively measured by using the parameter of two-dimensional cell area, with dedifferentiated cells exhibiting a larger area than the differentiated cells (3). We have previously reported that the mammalian target of rapamycin (mTOR) inhibitor rapamycin, which potently inhibits restenosis in vivo (18), stimulates VSMC differentiation in culture (16). As shown in Fig. 4, treatment of synthetic phenotype VSMC with 10 nmol/l iloprost for 48 h resulted in a differentiated morphology comparable to the effects of 20 nmol/l rapamycin, as indicated by a nearly 40% decrease in cell area relative to VSMC treated with vehicle control.


Figure 4
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  		Fig. 4. Iloprost induces a contractile morphology in VSMC. Bovine VSMC were cultured with ethanol vehicle, 10 nmol/l iloprost, or 20 nmol/l rapamycin for 48 h. VSMC two-dimensional (2D) cell area was determined in µm2 as described in MATERIALS AND METHODS. Error bars = SE; P values for Newman-Keuls multiple comparison post hoc test are indicated above the bars: ***P < 0.001 relative to control.

 
8-Br-cAMP mimics iloprost-induced VSMC differentiation. We next aimed to determine the signaling mechanism by which iloprost induces contractile protein expression. In Fig. 1, we demonstrated the concentration-dependent generation of cAMP by iloprost. Figure 5A shows that iloprost treatment similarly led to a concentration-dependent increase in PKA kinase activity, as measured using a nonradioactive in vitro PKA assay, with an EC50 of 30 nmol/l. In this assay, phosphorylation of the fluorescence-labeled PKA substrate kemptide results in an altered net charge (negative), thus allowing for the differential electrophoretic migration of the phosphorylated vs. the nonphosphorylated forms of the substrate. Addition of a specific PKA inhibitor peptide (PKI) to the in vitro reaction completely blocked the phosphorylation of the substrate, indicating that the kinase activity is PKA specific (data not shown). The substrate was not consumed to an extent greater than 25% of total for concentrations up to 10 nmol/l iloprost in Fig. 5A. We validated the kinase assay conditions by performing several important control experiments. Increasing concentrations of substrate with a fixed amount of enzyme (lysate) revealed Michaelis-Menten kinetics and verified that the 2-µg substrate used for all other experiments is well within the linear range of the assay (Fig. 5B). Furthermore, increasing concentrations of lysate with a fixed amount of substrate also showed that PKA activity is linear in proportion to the amount of lysate (enzyme) for the concentrations used in our experiments (25–30 µg lysate) (Fig. 5C).

We hypothesized that iloprost-stimulated PKA activation plays an important role in mediating the induction of VSMC differentiation markers. To test this hypothesis, we used the membrane-permeable cAMP analog 8-Br-cAMP to determine if this would mimic the effect of iloprost. As shown in Fig. 6, treatment with 500 nmol/l 8-Br-cAMP resulted in an increased expression of SM differentiation markers h-caldesmon (Fig. 6A) and SM2-MHC (Fig. 6B) that was similar to the effects of iloprost. Additionally, we have demonstrated that increasing concentrations of 8-Br-cAMP yield a concentration-dependent increase in PKA activation, as measured by using a nonradioactive in vitro PKA assay (Fig. 6C). From these data, we are able to conclude that comparable levels of PKA activation by either 8-Br-cAMP or iloprost promote VSMC differentiation.


Figure 6
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  		Fig. 6. cAMP analog induces contractile protein expression. A and B: human VSMC were stimulated with vehicle, 2.5 nmol/l iloprost, or with 500 nmol/l 8-bromo-cAMP (8-Br-cAMP) for 18 h. Cell lysates were analyzed by Western blotting by using antibodies against h-caldesmon (A) and SM2-MHC (B). Bar graph above shows densitometric quantitation from a single experiment in which each treatment group was performed in triplicate, with band intensities corrected to the loading control GAPDH. Results are expressed as means ± SE. C: cell lysates were prepared from human VSMC stimulated with increasing concentrations of 8-Br-cAMP for 15 min and analyzed for PKA activity by using a nonradioactive in vitro PKA assay. A representative experiment is shown.

 
PKA inhibition opposes iloprost induced VSMC differentiation. Because PKA activation was sufficient to induce VSMC differentiation, we next tested whether PKA is necessary for iloprost-induced differentiation. Using the PKA kinase inhibitors H-89 or KT-5720, we found that inhibition of PKA indeed inhibited iloprost-induced contractile protein expression (data not shown). However, as these pharmacological inhibitors are not entirely specific for PKA (6), we sought to inhibit PKA activity using more direct approaches. We employed two distinct, highly specific inhibitors of PKA function: the highly specific, cell-permeable (NH2 terminal myristoylated) protein kinase A inhibitor peptide (myrPKI), as well as siRNA targeted to PKA catalytic subunits. Treatment of human VSMC with 5 µmol/l myrPKI opposed the PKA activity induced by iloprost by 22%, as measured by the in vitro PKA kinase assay (Fig. 7A). This partial inhibition is likely because myrPKI is specifically membrane targeted and thus affects only a subset of total cellular PKA. This inhibition of PKA activity was accompanied by a complete repression of the iloprost-induced increase in the SM differentiation markers SM2-MHC, h-caldesmon, and calponin (Fig. 7, B–D).

Human VSMC have been shown to have a very low transfection efficiency, typically on the order of <1%. However, we have taken advantage of a new technology to efficiently and specifically knock down expression of the PKA catalytic subunits in human VSMC by using siRNA. There are two partially redundant isoforms of PKA catalytic subunits in VSMC, PKA-C{alpha} and -Cbeta; thus we designed siRNA selective for each subunit. Transfection of VSMC with PKA-C{alpha}- and PKA-Cbeta-specific siRNA resulted in a 60% knockdown of both PKA-C{alpha} and PKA-Cbeta mRNA levels after 30 h (Fig. 7F) and efficient knockdown of the corresponding protein levels 40 h posttransfection (data not shown). Basal PKA kinase activity was inhibited by 46–47% (Fig. 7E). The nontargeting siRNA control (siCONTROL) had no effect on the message levels of either subunit. None of the siRNAs had any effect on the levels of the housekeeping gene PDH (Fig. 7F). The resultant knockdown of PKA-C{alpha} and -Cbeta did, however, result in a nearly complete repression of the calponin and MHC induced by iloprost, as measured by semiquantitative RT-PCR (Fig. 7F). These data indicate that iloprost modulates VSMC phenotype via activation of the cAMP/PKA pathway.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Prostacyclin is an important endogenous regulator of vascular homeostasis. It plays an essential function in protecting the vasculature, including inhibiting platelet aggregation, promoting vasodilation, inhibiting leukocyte adhesion to endothelial cells, and inhibiting VSMC proliferation and migration (5, 7, 14, 20, 26). With its very short half-life, prostacyclin acts in a paracrine manner: PGI2 is predominantly synthesized via COX-2 in endothelial cells and released to act on neighboring VSMC (17). Thus endothelial dysfunction, which often occurs in vascular diseases such as restenosis and atherosclerosis, would reduce prostacyclin levels, which could potentially contribute to the diseased state. The recent withdrawal of the specific COX-2 inhibitors rofecoxib and valdecoxib as well as increasing reports of the dangers of several other members of this drug class have highlighted the cardioprotective role that prostacyclin plays in cardiovascular events (34). Selective COX-2 inhibitors reduce prostacyclin levels without affecting thromboxane levels, thus shifting the balance from vascular homeostasis toward prothrombotic conditions.

Another important contributing factor to the progression of both atherosclerotic and restenotic vascular disease is VSMC phenotypic modulation from a normal quiescent, contractile phenotype to a synthetic phenotype, whereby VSMC reenter the cell cycle, migrate into the intima, and secrete ECM (25, 28). Loss of contractile protein expression is a key feature of this synthetic phenotype. Although it is known that prostacyclin receptor signaling inhibits VSMC proliferation, it is well-established that in VSMC, unlike many other cell types, differentiation and proliferation are not coupled processes (25). Inhibition of proliferation does not obligately promote SMC differentiation (22, 24). Thus we hypothesized that in addition to inhibiting VSMC proliferation, prostacyclin might also protect the vasculature by maintaining VSMC in a differentiated state by actively inducing a set of SM-specific genes characteristic of the contractile phenotype. This would contribute to maintaining the contractile function but would also prevent the detrimental effects of dedifferentiation, such as lumen loss due to VSMC migration, proliferation, and matrix deposition. Here we report the novel observation that the prostacyclin mimetic iloprost modulates adult human VSMC from a synthetic to a contractile differentiated phenotype.

We then proceeded to determine the mechanism for the differentiated phenotype. We pursued the hypothesis that iloprost induces VSMC differentiation via activation of the downstream effector pathway Gs/AC/cAMP/PKA. We demonstrate that iloprost stimulates a rapid accumulation of cAMP and downstream PKA activity. Interestingly, it is known that cAMP triggers cellular differentiation in the pheochromocytoma cell line PC12 (4, 31). This cAMP-dependent neural cell differentiation is mediated by the induction of a set of specific genes via the activation of the transcription factor cAMP response element-binding protein (CREB) by PKA (31). Our study demonstrates that activation of PKA is also responsible for the iloprost-induced VSMC differentiation. Using a cAMP analog at concentrations that activate PKA to a similar extent as iloprost, we have shown that a cAMP signal is sufficient to induce expression of genes at the RNA level that characterize the differentiated phenotype. We are currently investigating PKA-regulated VMSC-specific transcription factors that may mediate this effect.

Using a variety of approaches to inhibit PKA activity, we have further shown that PKA activation is necessary for iloprost-induced VSMC differentiation. The specific inhibitor peptide myrPKI completely reversed the iloprost-induced expression of SM-specific differentiation markers. Interestingly, the basal levels of these markers were also decreased by myrPKI. Using RNA interference technology to knock down expression of the PKA catalytic subunits, we report that partial ablation of PKA expression was sufficient to completely prevent the iloprost-induced upregulation of SM-specific differentiation contractile markers. In contrast to the myrPKI approach, the basal levels of these markers were unaffected. Because both methods are highly specific for PKA, we found that while the siRNA knocked down both PKA mRNA and protein, kinase activity was only inhibited by 46–47%. This suggests that the residual PKA activity was likely sufficient to maintain basal levels of contractile protein expression.

An intriguing finding with the myrPKI peptide suggests that inhibition of membrane-associated PKA activity may be sufficient to inhibit both iloprost-induced and basal expression of contractile proteins. The NH2-terminal myristoylation motif on the PKI peptide is a means to increase cell permeability but notably targets the peptide to membranes. Our PKA kinase assays revealed that the myrPKI only inhibits total PKA activity by 22%, suggesting that it is indeed targeting a subset of PKA in the cell. The complete inhibition of contractile protein expression reveals the novel observation that a membrane-associated subcellular pool of PKA is responsible for the effects of iloprost. We are currently studying the mechanisms by which iloprost activates membrane-associated PKA and in turn regulates SM-specific differentiation markers.

In conclusion, our finding that prostacyclin receptor signaling induces VSMC differentiation via the cAMP/PKA pathway highlights another important role for prostacyclin in protecting the vasculature and provides a potential mechanism as to how differentiation is modulated under physiological conditions, and more importantly, how VSMC phenotype can be modulated under pathophysiological conditions such as restenosis, atherosclerosis, and blood vessel remodeling.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by grants from the National Heart, Lung, and Blood Institute to R. J. Powell, J. Hwa, and K. A. Martin and a National Cancer Institute training grant to K. M. Fetalvero.


   ACKNOWLEDGMENTS
 
We are grateful to Drs. David Brown, Erin Rowell, and Zsolt Kasza for assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. A. Martin, Section of Vascular Surgery, Dartmouth Medical School, Dartmouth-Hitchcock Medical Center, 1 Medical Center Dr., Lebanon, NH 03756 (e-mail: kathleen.a.martin{at}dartmouth.edu)

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
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Boger RH, BodeBoger SM, Thiele W, Junker W, Alexander K, and Frolich JC. Biochemical evidence for impaired nitric oxide synthesis in patients with peripheral arterial occlusive disease. Circulation 95: 2068–2074, 1997.[Abstract/Free Full Text]
  2. Boie Y, Rushmore TH, Darmongoodwin A, Grygorczyk R, Slipetz DM, Metters KM, and Abramovitz M. Cloning and expression of a cDNA for the human prostanoid IP receptor. J Biol Chem 269: 12173–12178, 1994.[Abstract/Free Full Text]
  3. Brown DJ, Rzucidlo EM, Merenick BL, Wagner RJ, Martin KA, and Powell RJ. Endothelial cell activation of the smooth muscle cell phosphoinositide 3-kinase/Akt pathway promotes differentiation. J Vasc Surg 41: 509–516, 2005.[CrossRef][Web of Science][Medline]
  4. Cassano S, Di Lieto A, Cerillo R, and Avvedimento EV. Membrane-bound cAMP-dependent protein kinase controls cAMP-induced differentiation in PC12 cells. J Biol Chem 274: 32574–32579, 1999.[Abstract/Free Full Text]
  5. Cheng Y, Austin SC, Rocca B, Koller BH, Coffman TM, Grosser T, Lawson JA, and FitzGerald GA. Role of prostacyclin in the cardiovascular response to thromboxane A2. Science 296: 539–541, 2002.[Abstract/Free Full Text]
  6. Davies SP, Reddy H, Caivano M, and Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351: 95–105, 2000.[CrossRef][Web of Science][Medline]
  7. Egan KM, Wang M, Lucitt MB, Zukas AM, Pure E, Lawson JA, and FitzGerald GA. Cyclooxygenases, thromboxane, and atherosclerosis-plaque destabilization by cyclooxygenase-2 inhibition combined with thromboxane receptor antagonism. Circulation 111: 334–342, 2005.[Abstract/Free Full Text]
  8. Feron O, Dessy C, Moniotte S, Desager JP, and Balligand JL. Hypercholesterolemia decreases nitric oxide production by promoting the interaction of caveolin and endothelial nitric oxide synthase. J Clin Invest 103: 897–905, 1999.[Web of Science][Medline]
  9. Fillinger MF, Sampson LN, Cronenwett JL, Powell RJ, and Wagner RJ. Coculture of endothelial cells and smooth muscle cells in bilayer and conditioned media models. J Surg Res 67: 169–178, 1997.[CrossRef][Web of Science][Medline]
  10. FitzGerald GA. Mechanisms of platelet activation—thromboxane A2 as an amplifying signal for other agonists. Am J Cardiol 68: B11–B15, 1991.[CrossRef][Medline]
  11. Gryglewski RJ, Botting RM, and Vane JR. Mediators produced by the endothelial cell. Hypertension 12: 530–548, 1988.[Abstract/Free Full Text]
  12. Hayashi K, Saga H, Chimori Y, Kimura K, Yamanaka Y, and Sobue K. Differentiated phenotype of smooth muscle cells depends on signaling pathways through insulin-like growth factors and phosphatidylinositol 3-kinase. J Biol Chem 273: 28860–28867, 1998.[Abstract/Free Full Text]
  13. Jaschonek K, Faul C, Schmidt H, and Renn W. Desensitization of platelets to iloprost-loss of specific binding-sites and heterologous desensitization of adenylate-cyclase. Eur J Pharmacol 147: 187–196, 1988.[CrossRef][Web of Science][Medline]
  14. Kobayashi T, Tahara Y, Matsumoto M, Iguchi M, Sano H, Murayama T, Arai H, Oida H, Yurugi-Kobayashi T, Yamashita JK, Katagiri H, Majima M, Yokode M, Kita T, and Narumiya S. Roles of thromboxane A2 and prostacyclin in the development of atherosclerosis in apoE-deficient mice. J Clin Invest 114: 784–794, 2004.[CrossRef][Web of Science][Medline]
  15. Kuro-o M, Nagai R, Tsuchimochi H, Katoh H, Yazaki Y, Ohkubo A, and Takaku F. Developmentally regulated expression of vascular smooth muscle myosin heavy-chain isoforms. J Biol Chem 264: 18272–18275, 1989.[Abstract/Free Full Text]
  16. Martin KA, Rzucidlo KM, Merenick BL, Fingar DC, Brown DJ, Wagner RJ, and Powell RJ. The mTOR/p70 S6K1 pathway regulates vascular smooth muscle cell differentiation. Am J Physiol Cell Physiol 286: C507–C517, 2004.[Abstract/Free Full Text]
  17. Mcadam BF, Catella-Lawson F, Mardini IA, Kapoor S, Lawson JA, and FitzGerald GA. Systemic biosynthesis of prostacyclin by cyclooxygenase (COX)-2: the human pharmacology of a selective inhibitor of COX-2. Proc Natl Acad Sci USA 96: 272–277, 1999.[Abstract/Free Full Text]
  18. Morice M, Serruys PW, Sousa JE, Fajadet J, Hayashi EB, Perin M, Colombo A, Schuler G, Barragan P, Guagliumi G, Molnar F, and Falotico R. A randomized comparison of a sirolimus-eluting stent with a standard stent for coronary revascularization. N Engl J Med 346: 1773–1780, 2002.[Abstract/Free Full Text]
  19. Munzel T, Daiber A, Ullrich V, and Mulsch A. Vascular consequences of endothelial nitric oxide synthase uncoupling for the activity and expression of the soluble guanylyl cyclase and the cGMP-dependent protein kinase. Arterioscler Thromb Vasc Biol 25: 1551–1557, 2005.[Abstract/Free Full Text]
  20. Murata T, Ushikubi F, Matsuoka T, Hirata M, Yamasaki A, Sugimoto Y, Ichikawa A, Aze Y, Tanaka T, Yoshida N, Ueno A, Ohishi S, and Narumiya S. Altered pain perception and inflammatory response in mice lacking prostacyclin receptor. Nature 388: 678–682, 1997.[CrossRef][Medline]
  21. Narumiya S, Sugimoto Y, and Ushikubi F. Prostanoid receptors: structures, properties, and functions. Physiol Rev 79: 1193–1226, 1999.[Abstract/Free Full Text]
  22. O'Brien ER, Alpers CE, Stewart DK, Ferguson M, Tran N, Gordon D, Benditt EP, Hinohara T, Simpson JB, and Schwartz SM. Proliferation in primary and restenotic coronary atherectomy tissue—implications for antiproliferative therapy. Circ Res 73: 223–231, 1993.[Abstract/Free Full Text]
  23. Owens GK. Regulation of differentiation of vascular smooth-muscle cells. Physiol Rev 75: 487–517, 1995.[Abstract/Free Full Text]
  24. Owens GK and Thompson MM. Developmental changes in isoactin expression in rat aortic smooth muscle cells in vivo—relationship between growth and cytodifferentiation. J Biol Chem 261: 3373–3380, 1986.
  25. Owens GK, Kumar MS, and Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev 84: 767–801, 2004.[Abstract/Free Full Text]
  26. Rudic RD, Brinster D, Cheng Y, Fries S, Song WL, Austin S, Coffman TM, and FitzGerald GA. COX-2-derived prostacyclin modulates vascular remodeling. Circ Res 96: 1240–1247, 2005.[Abstract/Free Full Text]
  27. Samuelsson B, Goldyne M, Granstrom E, Hamberg M, Hammarstrom S, and Malmsten C. Prostaglandins and thromboxanes. Annu Rev Biochem 47: 997–1029, 1978.[CrossRef][Web of Science][Medline]
  28. Sobue K, Hayashi K, and Nishida W. Expressional regulation of smooth muscle cell-specific genes in association with phenotypic modulation. Mol Cell Biochem 190: 105–118, 1999.[CrossRef][Web of Science][Medline]
  29. Stitham J, Stojanovic A, Ross LA, Blount AC, and Hwa J. Clusters of transmembrane residues are critical for human prostacyclin receptor activation. Biochemistry 43: 8974–8986, 2004.[CrossRef][Medline]
  30. Stork PJS and Schmitt JM. Crosstalk between cAMP and MAP kinase signaling in the regulation of cell proliferation. Trends Cell Biol 12: 258–266, 2002.[CrossRef][Web of Science][Medline]
  31. Vane J and Corin RE. Prostacyclin: a vascular mediator. Eur J Vasc Endovasc Surg 26: 571–578, 2003.[CrossRef][Web of Science][Medline]
  32. Vane JR, Anggard EE, and Botting RM. Mechanisms of disease—regulatory functions of the vascular endothelium. N Engl J Med 323: 27–36, 1990.[Web of Science][Medline]
  33. Wong DR, Wang MA, Cheng Y, and FitzGerald GA. Cardiovascular hazard and non-steroidal anti-inflammatory drugs. Curr Opin Pharmacol 5: 204–210, 2005.[CrossRef][Web of Science][Medline]



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