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Circulation Research. 2004;94:592-600
Published online before print January 29, 2004, doi: 10.1161/01.RES.0000119169.87429.A0
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(Circulation Research. 2004;94:592.)
© 2004 American Heart Association, Inc.

Molecular Medicine

Induction of Hyaluronic Acid Synthase 2 (HAS2) in Human Vascular Smooth Muscle Cells by Vasodilatory Prostaglandins

M. Sussmann, M. Sarbia, J. Meyer-Kirchrath, R.M. Nüsing, K. Schrör, J.W. Fischer

From the Institut für Pharmakologie und Klinische Pharmakologie (M. Sussmann, J.M.-K., K.S., J.W.F.), Heinrich Heine Universität Düsseldorf; Institut für Allgemeine Pathologie der Technischen Universität München (M. Sarbia), München; Klinik für Allgemeine Kinderheilkunde (R.M.N.), Phillips Universität, Marburg, Germany.

Correspondence to Jens W. Fischer, Molecular Pharmacology, Institut für Pharmakologie und Klinische Pharmakologie, Heinrich-Heine-Universität Düsseldorf, Moorenstrasse 5, D-40225 Düsseldorf, Germany. E-mail jens.fischer{at}uni-duesseldorf.de


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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Hyaluronic acid (HA) is a prominent constituent of the extracellular matrix of atherosclerotic vascular lesions in humans known to modulate vascular smooth muscle phenotype. The regulation of HA synthesis by vasodilatory prostaglandins was analyzed in human arterial smooth muscle cells (SMCs). The prostacyclin analogue, iloprost (100 nmol/L), markedly increased pericellular formation of HA coats and HA secretion into the cell culture medium in human arterial SMCs (8.7±1.6-fold). Expression of HA synthase 2 (HAS2) was determined by semiquantitative RT-PCR and found to be strongly upregulated at concentrations of iloprost between 1 and 100 nmol/L after 3 hours. Furthermore, endogenous cyclooxygenase-2 (COX2) activity was required for basal expression of HAS2 mRNA in SMCs in vitro. Total HA secretion in response to iloprost was markedly decreased by RNA interference (RNAi), specific for HAS2. In addition, siRNA targeting HAS2 strongly increased the spreading of human SMCs compared with mock-transfected cells. HAS2 mRNA levels were also stimulated by a selective prostacyclin receptor (IP) agonist, cicaprost (10 nmol/L), prostaglandin E2 (10 nmol/L), and the EP2 receptor agonist, butaprost (1 µmol/L). Induction of HAS2 mRNA and HA synthesis by prostaglandins was mimicked by stable cAMP analogues and forskolin. In human atherectomy specimens from the internal carotid artery, HA deposits and COX2 expression colocalized frequently. In addition, strong EP2 receptor expression was detected in SMCs in HA-rich areas. Therefore, upregulation of HAS2 expression via EP2 and IP receptors might contribute to the accumulation of HA during human atherosclerosis, thereby mediating proatherosclerotic functions of COX2.


Key Words: hyaluronic acid • extracellular matrix • prostaglandins • cyclooxygenase-2 • atherosclerosis


*    Introduction
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 up arrowAbstract
 *Introduction
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Hyaluronic acid (HA) is a nonsulfated acidic carbohydrate of the extracellular matrix (ECM) consisting of repeating disaccharide units (D-glucuronic acid ß-1,3-N-acetylglucosamine-ß1,4). The molecular weight of native HA polymers ranges from 105 to 107 Da.1 In mammals, HA is synthesized by three HA synthase (HAS) isoforms: HAS1,2 HAS2,3,4 and HAS3.5 HAS are located at the inner surface of the cell membrane and extrude the growing HA polymer into the extracellular space. After dissociation from the HAS enzyme, HA interacts specifically with the cell surface receptors CD44 and RHAMM (receptor of HA-mediated motility) as well as with a variety of secreted proteins (hyaladherins) such as link protein, versican, and TSG-6 (tumor necrosis factor–induced gene-66). These HA/protein interactions allow the formation of large ECM networks that also interweave other ECM molecules and form the microenvironment of vascular smooth muscle cells (SMCs).

HA-rich matrices are essential for cell migration and proliferation during embryonic development and organogenesis.7 Furthermore, HA-rich matrices are characteristic for many tumors and seem to be critically involved in tumor cell migration and invasion.8 Similarly, in SMC HA-rich matrices are thought to promote the proliferative and migratory phenotype.9,10 HA is a common constituent of restenotic and atherosclerotic lesions both in animal models and in humans.11 However, information about the regulatory pathways that govern HA synthesis in SMCs during atherosclerosis is still very limited. It has been noted that platelet-derived growth factor-BB (PDGF-BB)12 and transforming growth factor-ß (TGF-ß)13 induce HA synthesis in SMCs.

During the progression of atherosclerosis SMCs are exposed to locally generated prostaglandins, because cyclooxygenase-2 (COX2) expression and subsequent prostacyclin- and prostaglandin E (PGE) synthesis are strongly elevated in macrophages and SMCs of atherosclerotic lesions.14–17 Recently, we have found that HAS2 is among the 51 genes that are induced by the prostacyclin (PGI2) analogue, iloprost, in human arterial SMCs.18 Further hints that vasodilatory prostaglandins might be considered regulators of HA synthesis in SMCs have been provided by studies in fibroblasts and pericardial mesothelial cells showing that PGE2 stimulates HA synthesis.19,20

Therefore, the aim of the present study was to investigate (1) whether vasodilatory prostaglandins such as PGI2 and PGE2 regulate HA synthesis in human SMCs, (2) to identify the responsible prostaglandin receptors and signal transduction pathways, and (3) to determine the spatial relationship of HA, COX2, and prostaglandin receptors in human atherosclerotic plaques.


*    Materials and Methods
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 *Materials and Methods
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Reagents
Iloprost and cicaprost were kindly provided by Schering AG. Forskolin, prostaglandin E2 (PGE2), dibutyryl-cAMP (db-cAMP), PDGF-BB, H89, pertussis toxin (PTX), rottlerin, and GF109203X were purchased from Sigma. SC19220 was obtained from Tocris, butaprost was kindly provided by Dr P. Gardiner, Bayer (Middlesex, UK), ONO-AE1-329 by ONO Pharmaceuticals (Osaka, Japan), and M&B28.767 by Dr L. Caton, Rhône-Poulenc Rorer (Virtry sur Seine, France). Calphostin C was from Calbiochem. Etoricoxib was obtained from Laboratorien Berlin Aldersdorf GmbH. All cell culture reagents were from Invitrogen. Polyclonal COX2 antibody (H62) for Western blotting was obtained from Santa Cruz.

Cell Culture
Arterial SMCs were isolated by the explant technique from the media of human internal mammary arteries. Leftover fragments were obtained from patients undergoing aortocoronary bypass surgery according to the guidelines of the local Medical Ethical Board. SMCs of passages 4 to 10 were used. Three to four different arterial cell lines were studied. The SMCs were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 15% FCS (fetal calf serum), 100 U/mL penicillin, and 100 µg/mL streptomycin in a humidified atmosphere with 5% CO2 at 37°C. Cells were growth-arrested by serum withdrawal for 24 hours and were subsequently treated with the compounds to be studied.

Quantitation of HA
The amount of HA in the cell culture supernatant was measured by a radiometric assay (Pharmacia HA Test, Pharmacia-Diagnostics). This assay is based on the specific interaction of HA with the radio-labeled HA-binding protein (HABP-[125I]) from bovine cartilage. The quantitation of HA was performed in duplicates according to the instructions of the manufacturer using 100 µL aliquots of the conditioned cell culture medium.

Quantitation of 6-Keto-PGF1{alpha}
Cell culture supernatants were collected at the indicated times, and the concentration of 6-keto-PGF1{alpha}, the stable hydrolysis product of PGI2, was determined by RIA as described previously.21

Detection of Pericellular HA Coats
Pericellular matrices were visualized using a particle exclusion assay as described previously.22 Briefly, human arterial SMCs were cultured in 6-well plates under normal growth conditions as defined above. Cells were growth-arrested for 24 hours by serum withdrawal and subsequently stimulated with iloprost for 24 hours. Then, 750 µL of 1% paraformaldehyde-fixed, washed human erythrocytes (108/mL) were added to the cell-layer and incubated for 15 minutes. Pericellular coats were visualized by exclusion of sedimented erythrocytes.

Morphometric Analysis of Cultured SMCs
Cell area was determined using analySIS software (Soft Imaging System, Germany) in 6-well dishes. For this purpose, three images of each experimental setup were taken randomly (x100 original magnification), and the area of substratum covered by each SMC measured (40 to 60 cells per condition).

RNA Isolation
Total RNA from arterial SMCs was isolated by using peqGOLD Trifast (PeqLab) following the manufacturer’s manual. The RNA was quantitated by spectroscopic analysis at 260 nm.

Determination of HAS2 Expression
The expression levels of HAS2 were analyzed by semiquantitative RT-PCR (One-Step RT-PCR kit; Qiagen) using 250 ng of total mRNA for each RT-PCR reaction. The following oligonucleotides (Invitrogen) were used: HAS2 (forward, 5'-GTCTCAAATTCATCTGATCTC-3'; reverse, 5'-ACATTTCCTTAAGTAGTCTGG-3'), product size: 419 bp; glycerol-aldehyde-3-phosphate-dehydrogenase (GAPDH) (forward, 5'-TGATGACATCAAGAAGGTGGTTGAA-3'; reverse, 5'-TCCT-TGGAGGCCATGTAGGCCAT-3'), product size: 219 bp. GAPDH was coamplified as reference gene to allow semiquantitative evaluation of the expression levels. After reverse transcription (30 minutes, 50°C) and denaturation of DNA (15 minutes, 95°C), the following thermal profile was performed: HAS2: 28 cycles of 1 minute 95°C, 1 minute 51°C and 1 minute 72°C and a final elongation step at 72°C for 10 minutes. Subsequently, RT-PCR products were run on a 2% agarose gel, stained with ethidium bromide and fluorescent bands quantitated using Quantity One 4.1-1 (Biorad). The expression level was calculated as ratio of the respective HAS2 and GAPDH bands and normalized to the unstimulated control.

Transfection of Vascular SMCs With siRNA
Single-stranded siRNAs, with 19-nt duplex RNA and 2-nt 3'dTdT overhangs were synthesized by Qiagen-Xeragon. The siRNA sequences targeting HAS2 were 5'-UUGGAACCACACUCUUUGGd(-TT)-3' and 5'-CCAAAGAGUGUGGUUCCUUd(TT)-3'. Cultured arterial SMCs were trypsinized, seeded at a cell density of 50 000 cells per 12-well (3.5 cm2), and cultured in DMEM with 15% FCS and antibiotics as described earlier. After 24 hours, SMCs were serum-deprived and transfected with siRNA (1.6 µg/well) using the TransMessenger Transfection Reagent (Qiagen) according to the instructions of the manufacturer. Cells were kept serum-free for an additional 24 hours before the experiments. HAS expression or HA synthesis was studied as indicated.

Analysis of Human Atherosclerotic Lesions
Atherectomy specimens from the internal carotid artery [9 females, 10 males; median age 68 years (range, 52 to 83)] were collected retrospectively from the files of the Institute of Pathology, University of Düsseldorf, Germany. All cases had been operated for symptomatic occlusive disease of one of the internal carotid arteries. The specimens were fixed in 4% buffered formaldehyde, cut transversally, and embedded in paraffin. HA was detected using HABP (Calbiochem; 1:100) as described previously.23,24 In addition, antibodies against COX1 (monoclonal, CXIII, 1:100, Alexis), COX2 (polyclonal, 1:150, Cayman Chemical Company), CD68 (monoclonal, PG-M1, 1:500, DAKO), SM-actin (monoclonal, HHF 35, 1:100, DAKO), and EP2 receptor (polyclonal,25 1:100). Double staining was performed detecting first HABP with a streptavidin/horseradish peroxidase–coupled secondary antibody and diaminobenzidine (DAKO) as a chromogen. Subsequently, the slides were incubated with one of the above mentioned primary antibodies (1 hour; room temperature). The primary antibodies were then detected with an alkaline phosphatase (AP)–conjugated secondary antibody and the reaction was developed using Fast Red (DAKO) as chromogen. Finally, the slides were counterstained with hemalaun. The staining patterns were evaluated by a senior pathologist (M. Sarbia.).

Statistical Analysis
Data are the mean±SD of n independent experiments. Statistical analysis was performed by one-way ANOVA followed by Dunnett’s multiple comparison test or comparison of selected pairs (Bonferroni). A value of P<0.05 was considered significant.


*    Results
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 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
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Induction of Pericellular HA Coats and HA Synthesis by Iloprost
HA coats were markedly enlarged at 24 hours in response to iloprost as shown in Figure 1A. Subsequently, the HA concentration in the cell culture supernatant was measured. Within 24 hours, approximately 40 ng HA/10 000 cells (range in n=3 different cell lines: 20 to 70 ng HA/10 000 cells) were secreted by serum-starved SMCs. Because the absolute amounts of HA secreted by SMCs varied substantially between various cell lines, data are expressed as fold increase above controls. Iloprost caused an approximately 9-fold increase of secreted HA after 24 hours (Figure 1B). This induction of HA accumulation by iloprost was comparable to that observed at high concentrations of PDGF-BB (20 ng/mL; 6.6±3.8-fold over control).



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  		Figure 1. A, Pericellular HA coat formation in SMCs visualized by exclusion of sedimented erythrocytes using light microscopy; a and b, control; c and d, 100 nmol/L iloprost; original magnifications were x40 (a and c) and x200 (b and d); black arrowheads, plasma membrane; white arrows, outer border of pericellular ECM coat. B, HA concentration measured in the cell culture medium 24 hours after stimulation with iloprost or PDGF-BB. Data are normalized to the respective controls; n=3, mean±SD. *P<0.05 vs control.

Time- and Concentration-Dependent Induction of HAS2 by Iloprost
The effect of iloprost on mRNA expression of HAS2, the major HAS isoform in SMCs,10 was determined by RT-PCR. To accomplish this, 1, 3, 6, 16, and 24 hours after addition of iloprost, cells were harvested, total mRNA was isolated, and semiquantitative RT-PCR for HAS2 was performed (Figure 2A). Maximum mRNA levels were reached 3 and 6 hours after addition of iloprost as previously described.18 The average induction of HAS2 mRNA at 3 hours was 3.5±1.4-fold above controls (n=6). Based on these results, in subsequent experiments, we determined HAS2 mRNA levels at 3 hours, if not indicated otherwise. As observed with respect to HA accumulation, concentrations of iloprost as low as 1 nmol/L strongly elevated HAS2 mRNA in arterial SMCs (Figure 2B). In addition, the mRNA induction was in the same order of magnitude as found for PDGF-BB (20 ng/mL; data not shown). These results demonstrate that iloprost strongly induces HA secretion and HA coat formation probably via induction of HAS2 mRNA expression.



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  		Figure 2. A, Time course of HAS2 mRNA induction in response to iloprost (100 nmol/L) in arterial SMCs; semiquantitative RT-PCR. After densitometry, the ratio of the signals of HAS2 and GAPDH were normalized to control. B, Concentration dependency of HAS2 mRNA induction in response to iloprost; total RNA was isolated 3 hours after addition of iloprost. Inset shows the results of a representative RT-PCR. In A and B, data of n=3 and n=6 (100 nmol/L iloprost, 3 hours) independent experiments are shown; mean±SD. *P<0.05 vs control.

HAS2 Expression Is Dependent on Endogenous COX2
After 12-hour incubation of serum-starved SMCs with etoricoxib (10 µmol/L), a specific inhibitor of COX2, expression of HAS2 mRNA was almost undetectable (Figure 3A). In contrast, untreated, starved control cells showed basal HAS2 mRNA expression. Expression of COX2 in these cells was verified by Western analysis (Figure 3B). Furthermore, 6-keto-PGF1{alpha}, the stable metabolite of PGI2, was determined in the supernatant of serum-starved control cells. Within 24 hours, a concentration of 2.7±0.42 nmol/L 6-keto-PGF1{alpha} (n=4) was found in the cell culture supernatant. Thus, the concentrations of endogenous PGI2 are well within the range of concentrations that strongly induce HA secretion and HAS2 mRNA expression (Figures 1 and 2Up).



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  		Figure 3. A, HAS2 mRNA expression in serum-starved (36 hours) SMCs in the absence and presence of 10 µmol/L etoricoxib (added for the last 12 hours). Shown is the result of a representative RT-PCR and the densitometric quantitation of n=3 experiments. Mean±SD. *P<0.05 vs control. B, Western blot in SMCs treated as in A showing a typical double band for COX2 at 71 kDa.

siRNA Targeting HAS2 Inhibits Spreading of SMCs
Small interfering RNA duplexes (siRNA) targeting HAS2 were designed and used to transfect human arterial SMCs. Figure 4A shows that iloprost-induced HAS2 mRNA levels were markedly decreased in SMCs by this approach. The decrease in mRNA levels caused by siRNA corresponded to about 50% reduction of HA secretion (Figure 4B). Morphometric analysis of SMCs transfected with siRNA targeting HAS2 (Figure 4C) revealed that HAS2 participates in the regulation of cell shape and spreading. Suppression of HAS2 expression dramatically increased the substratum areas occupied by individual SMCs. The cytoplasma of the cells transfected with HAS2-siRNA appeared very thin and in close contact with the surface underneath (Figure 4D). The difference in spreading was not observed within the first 12 hours but was clearly detectable 20 hours after addition of iloprost to mock-transfected or HAS2-siRNA–transfected cells.



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  		Figure 4. A, HAS2 mRNA levels of unstimulated SMCs, mock-transfected SMCs, and SMCs transfected with HAS2-siRNA 3 hours after stimulation with 100 nmol/L iloprost; result of a representative experiment. B, Effect of HAS2-siRNA on HA secretion in response to iloprost (100 nmol/L). Levels of HA in the cell culture medium were determined 24 hours after treatment of cells as in A; representative experiment. C, Mean area occupied by control, mock-, and HAS2-siRNA–transfected cells 20 hours after addition of iloprost 100 nmol/L; n=4 independent experiments; mean±SD. *P<0.05 mock-transfected vs HAS2 siRNA. D, Representative interference-contrast images of SMCs treated as described in C; 200-fold original magnification.

Iloprost Induces HAS2 via Stimulation of IP Receptors
Iloprost is not only a ligand of IP receptors but also of prostaglandin E1 (EP1) receptors.26,27 Furthermore, human SMCs are known to express IP-receptors and a variety of EP receptors.28 Accordingly, mRNA transcripts of the IP, EP1, EP2, EP3, and EP4 receptors were detected by RT-PCR in SMCs from the mammary artery that were used in the present study (not shown). To identify the prostaglandin receptor subtype mediating the effect of iloprost on HAS2 mRNA expression, a selective IP receptor agonist and a selective EP1 receptor antagonist were used. Cicaprost, the specific IP receptor agonist29 strongly induced HAS2 mRNA levels and HA synthesis (Figures 5A and 5C). In contrast, the EP1 receptor antagonist SC1922030 (10 µmol/L) had no effect on iloprost induced HAS2 mRNA expression.



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  		Figure 5. A, Iloprost-induced HAS2 mRNA expression was mimicked by cicaprost (10 nmol/L); SC19220 (10 µmol/L) had no effect and H89 (100 nmol/L) inhibited HAS2 induction by iloprost (100 nmol/L). B, PGE2-induced (10 nmol/L) HAS2 mRNA expression was not affected by PTX (10 ng/mL) and mimicked by butaprost (1 µmol/L). M&B 28.7267 (100 nmol/L) and Ono-AE1-329 (100 nmol/L) had no effect. HAS2 mRNA expression was determined by semiquantitative RT-PCR after 3 hours in A and B. C, HA secretion over 24 hours after stimulation by cicaprost (100 nmol/L), PGE2 (100 mmol/L), and butaprost (1 µmol/L). Insets in A and B show results of representative experiments. Results are fold induction above unstimulated SMCs; in A, n=3 to 11; in B, n=3 to 4, iloprost n=2; in C, n=3 to 8; mean±SD. *P<0.05 vs control.

Prostaglandin E2 Induces HAS2 via EP2 Receptors
PGE2, a nonselective ligand of all EP receptors (EP1–4),29 induced HAS2 mRNA and HA synthesis as well (Figures 5B and 5C). The EP2 agonist butaprost (1 µmol/L)31 strongly induced HAS2 transcript levels and HA synthesis (Figures 5B and 5C). Involvement of the Gi-coupled EP3 receptor was excluded because pertussis toxin (10 ng/mL) did not alter HAS2 expression in response to PGE2. Moreover, M&B 28.767 (100 nmol/L), an EP3 agonist,32 had no effect. The EP4 receptor was not involved in HAS2 regulation, because the EP4 agonist Ono-AE1-329 (100 nmol/L) did not affect HAS2 mRNA expression (Figure 5B). The basal HAS2 mRNA expression was particularly low in the cell line used for the experiments shown in Figure 5B leading to a higher than average induction of HAS2 mRNA. However, the effects of PGE2 and butaprost were in the same order of magnitude as the effect of iloprost, which was applied in parallel (last column on the right) as internal control.

Involvement of PKA in HAS2 Induction by Vasodilatory Prostaglandins
IP and EP2 receptors are known to be coupled to stimulatory G-proteins and to initiate cAMP (cAMP)/protein kinase A (PKA)–dependent signal transduction pathways.33 Accordingly, we found that the PKA-inhibitor H89 (100 nmol/L) reduced the induction of HAS2 mRNA and HA synthesis by iloprost (Figure 5A) and butaprost (not shown). Moreover, db-cAMP (1 mmol/L) and forskolin (10 µmol/L) induced both HAS2 mRNA and HA secretion (Figures 6A and 6B). PKC-mediated signals are not involved in the regulation of HAS-2 by prostaglandins, because the PKC-inhibitors calphostin C (50 nmol/L), rottlerin (250 nmol/L), and GF109203X (100 nmol/L) had no effect (data not shown).



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  		Figure 6. A, HAS2 mRNA expression in response to db-cAMP (1 mmol/L) and forskolin (10 µmol/L). B, HA secretion into the culture medium (24 hours) in response to db-cAMP (1 mmol/L) and forskolin (10 µmol/L); n=3 to 11. *P<0.05 vs control.

Localization of HA and COX2 in Human Atherosclerotic Lesions
To investigate the significance of these findings in human atherosclerosis, atherectomy specimens were stained for HA, COX2, COX1, and EP2 receptors. All analyzed specimens contained multiple areas in the neointima that were strongly positive for HA. Frequently, COX2 was also detected at these HA-positive sites. Figure 7A shows a HA-positive area of the neointima. At higher magnification (inset a) the extracellular nature of the HA staining can be appreciated. The corresponding area (Figure 7C) in the consecutive section revealed strong cellular (inset c) staining for COX2. Another example for this colocalization is shown in Figures 7B and 7D in the neointima of a different individual. The arrow heads point to areas of strong HA and COX2 staining. However, it should be noted that HA was also observed in the absence of COX2 and vice versa (asterisks in Figure 7). In addition, double staining revealed that HA-rich areas contained both SM-actin–positive (SMC) cells and often also CD68-positive cells (macrophages) and that both cell types were positive for COX2 (not shown). Importantly, Figure 8 demonstrates that SM-actin–positive cells in HA-rich areas strongly express the EP2 receptor. In contrast, COX1 was detected in the endothelium but only very rarely in the neointima of the same lesions that strongly expressed COX2 and the EP2 receptor (data not shown).



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  		Figure 7. A and B, HA staining in atherectomy specimens of human internal carotid arteries using HABP as a specific probe. C and D, COX2 immunostaining of the corresponding areas in consecutive sections. Arrowheads, examples of areas positive for HA and COX2; asterisks, examples of areas with strong COX2 and weak HA staining. Original magnifications x100; insets a and c were photographed at x400.



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  		Figure 8. A, Double staining of HA (brown) and EP2 receptors (red) in an SMC-rich area of the neointima of human atherosclerotic plaques derived from carotid artery as shown in Figure 7. B, Staining of SM-actin (red) and of CD68 (red) (C) in consecutive sections; x400 original magnification.


*    Discussion
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 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
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The composition of ECM determines the volume of the neointima, the stability of fibrous caps, the thrombogenic properties, lipid accumulation, and finally the phenotype of the cells within the atherosclerotic lesion.34 The products of COX2 and PGES, which are expressed by macrophages and SMCs within atherosclerotic plaques, are thought to contribute to the inflammatory response, to destabilize atherosclerotic plaques via increased ECM degradation by matrix metalloproteinases, and to impair endothelial function.16,35,36 So far, nothing is known about regulation of HA synthesis in vascular SMCs by vasodilatory prostaglandins such as PGI2 and PGE2.

The data of this study reveal that agonists of IP receptors and EP2 receptors markedly induce pericellular coat formation and HA secretion in human SMCs at physiologically and pharmacologically relevant concentrations37 of the respective ligands. The induction of HA synthesis by IP and EP2 receptor ligands was comparable to the induction of HA synthesis by PDGF-BB, which has been described earlier to be a potent inducer of HA synthesis in SMCs.10

The present data revealed that human SMCs strongly upregulate HAS2 transcripts in response to iloprost and PGE2. The HAS2 mRNA induction was maximal at 3 and 6 hours after addition of iloprost and PGE2 and was followed by a rise of HA levels in the culture supernatant. The finding that inhibition of COX2 abrogated basal HAS2 expression in vitro suggested that this regulatory pathway might be physiologically relevant. Transfection of SMCs with specific HAS2 siRNAs decreased HAS2 mRNA levels and HA secretion in response to iloprost. These findings strongly suggest that elevation of HAS2 mRNA mediates prostaglandin induced HA accumulation. The HAS2 gene has been demonstrated to be the major HAS2 isoform in vascular SMCs10 and the primary target among the HAS isoforms for regulation of HA synthesis in other cell types. A variety of stimuli, such as EGF in keratinocytes,38 interleukin-1 in orbital fibroblasts,39 or PDGF-BB in mesothelial cells40 induced specifically HAS2 mRNA.

As both the IP and EP2 receptor are known to couple to stimulatory G-proteins and subsequently evoke a variety of effects by initiation of cAMP/PKA-dependent signal transduction, it was investigated whether HAS2 mRNA expression in human SMCs was induced by the cAMP/PKA pathway. This was the case, because both HA secretion and HAS2 mRNA were elevated by db-cAMP and forskolin and could be inhibited by H89, an inhibitor of PKA. These findings are in line with a previous report demonstrating cAMP-dependent induction of HA synthesis in response to PGE2 in pericardial mesothelial cells.19

In summary to this point, it was demonstrated in this study for the first time that IP and EP2 receptor agonists are potent inducers of HA synthesis via induction of HAS2 mRNA in a cAMP/PKA-dependent fashion in human arterial SMCs.

What are the pathobiological implications of these findings? HAS2 expression in response to iloprost appeared to be involved in the control of SMC shape and spreading, which is thought to strongly influence cell phenotype.41 In keratinocytes, it was recently shown that HAS2 antisense increased focal adhesion plaques and reduced migration and proliferation.42 In the present study, a similar phenotype was induced in SMCs, because spreading of SMCs was increased by HAS2-siRNA, suggesting that SMCs establish close contacts with the substratum in the absence of endogenous HAS2 expression and might have a reduced propensity to migrate and proliferate. This view is also supported by the literature on HAS2-mediated effects in SMCs. For example, HA-rich matrix is strongly induced by PDGF-BB in SMCs in vitro and likely supports the proliferative response to PDGF-BB, because interference with the binding of HA to its receptors CD44 and RHAMM by means of competing HA oligosaccharides9 or blocking antibodies to RHAMM12 inhibited SMC proliferation and migration. Therefore, it is likely that HA-rich matrix promotes the proliferative, migratory phenotype of SMCs. In accordance with this hypothesis, HA has also been found to associate with proliferating SMCs in experimental models of restenosis and atherosclerosis.43 Moreover, HA is a component of all stages of atherosclerotic lesions in humans.11,44 Next to phenotypic modulation and ECM expansion, HA might be involved in the recruitment of macrophages, because macrophages via CD44 receptors bind to HA on the endothelial surface and to HA within the arterial wall.45,46 Moreover, macrophage-containing areas appear to be enriched in HA in human atherosclerotic lesions.44,47

Several studies suggest that COX2, which is strongly induced in SMCs and macrophages during atherosclerosis, might aggravate the course of the disease. For example, in LDL-receptor knockout mice, genetic deletion or pharmacological inhibition of COX2 reduced atherosclerotic lesion formation.35 Furthermore, in humans, COX2 inhibition was recently shown to improve endothelial function and to decrease C-reactive protein and oxidized LDL.36 The results of the present study suggest that HAS2 induction and formation of HA-rich matrix might be part of the lesion-promoting, proinflammatory function of COX2-derived prostaglandins. This hypothesis is supported by the current analysis of atherectomy specimens derived from patients suffering from symptomatic occlusive disease of the internal carotid artery showing that (1) COX2-positive SMCs and macrophages were frequently embedded in HA-rich ECM and (2) that SMCs in the HA-rich ECM strongly express the EP2 receptor. It is thought that significant PGE2 levels can be generated within the lesions, because PGES is induced by proinflammatory stimuli48 and colocalizes with COX2 in the shoulder regions of plaques.16 Furthermore, it is likely, that HA synthesis in atherosclerotic lesions is dependent not only on the expression levels of COX1 and COX2, providing PGI2 synthase and PGE synthase with their substrate, PGH2, but also on the expression levels of IP and EP2 receptors. Because EP2 receptors were strongly expressed on SMCs surrounded by HA, PGE2 synthesized by COX2/PGES might be a likely candidate to induce HA synthesis in atherosclerotic lesions.

In conclusion, the present finding that in arterial SMCs, EP2 and IP receptor ligands induce HAS2 expression and synthesis of HA adds a new aspect to the ongoing debate about the role of COX2 in the progression of atherosclerosis.


*    Acknowledgments
 
This study was in part supported by the Forschungskommission (Universitätsklinikum Düsseldorf) and the Deutsche Forschungsgemeinschaft (SFB 612, B7). We thank Peggy Mann for excellent technical assistance.


*    Footnotes
 
Original received August 18, 2003; resubmission received December 2, 2003; revised resubmission received January 14, 2004; accepted January 16, 2004.


*    References
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 up arrowMaterials and Methods
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 *References
 

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