This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sachinidis, A. Articles by Vetter, H. Search for Related Content PubMed PubMed Citation Articles by Sachinidis, A. Articles by Vetter, H. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB CALCIUM COMPOUNDS CALCIUM, ELEMENTAL

(Hypertension. 1995;26:771.)
© 1995 American Heart Association, Inc.

Articles

Thromboxane A₂ and Vascular Smooth Muscle Cell Proliferation

Agapios Sachinidis; Markus Flesch; Yon Ko; Karsten Schrör; Michael Böhm; Rainer Düsing; Hans Vetter

From the Medizinische Universitäts-Poliklinik, Bonn (A.S., M.F., Y.K., R.D., H.V.); Institut für Pharmakologie, Düsseldorf (K.S.); and Klinik III für Innere Medizin der Universität zu Köln (M.B.) (Germany).

Correspondence to PD Dr A. Sachinidis, Medizinische Universitäts-Poliklinik, Wilhelmstr. 35-37, 53111 Bonn, FRG.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Abstract In the present study we describe the intracellular pathways for the transmission of growth signals by the potent vasoconstricting eicosanoids prostaglandin H₂ and thromboxane A₂ in smooth muscle cells from rat aorta. Carbocyclic thromboxane A₂ and U46619 are stable thromboxane A₂ mimetics acting at the common thromboxane A₂/prostaglandin H₂ receptor. Carbocyclic thromboxane A₂ (10^-6 mol/L) induced an approximately 2.5-fold increase in [Ca²⁺]_i above the basal value at 25 seconds. Maximal stimulation of the 42-kD mitogen-activated protein kinase isoform by both thromboxane A₂ mimetics occurred at 5 minutes. Both thromboxane A₂ mimetics at a concentration of 10^-6 mol/L induced the expression of c-fos and early growth response gene-1 (egr-1) mRNA, with a maximum at 30 minutes. Carbocyclic thromboxane A₂ (10^-6 mol/L) induced a 3.3-fold increase in [³H]thymidine incorporation into cell DNA above the basal value and produced a 3.5-fold elevation of platelet-derived growth factor-BB–dependent [³H]thymidine incorporation into cell DNA. Similar effects of U46619 (10^-6 to 10^-5 mol/L) alone and in combination with platelet-derived growth factor-BB on cell DNA synthesis were obtained. The thromboxane A₂/prostaglandin H₂ receptor antagonist SQ29548 (10^-6 mol/L) completely suppressed the mitogenic effect of both thromboxane A₂ mimetics (10^-6 mol/L). Pertussis toxin (10 to 100 ng/mL) did not influence the mitogenic effects of the thromboxane A₂ mimetics. Carbocyclic thromboxane A₂ (10^-6 mol/L) and platelet-derived growth factor-BB (20 ng/mL) per se caused a 44% and 100% increase in cell number, respectively. In the presence of carbocyclic thromboxane A₂ (10^-6 mol/L), platelet-derived growth factor-BB induced a 152% increase in cell number. Similar results were obtained with U46619 alone or in combination with platelet-derived growth factor-BB.

Key Words: thromboxane A₂ • RNA, messenger • calmodulin-dependent protein kinases • muscle, smooth, vascular • pertussis toxins

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
VSMC hypertrophy and proliferation may participate in the pathophysiology of both hypertension and atherosclerosis.^{1 2} TXA₂ and its immediate prostaglandin endoperoxide precursor PGH₂ are lipid metabolites of arachidonic acid that are synthesized from activated platelets and vessel wall tissues. They have been described as potent vasoconstrictor agents for VSMCs acting through a common pharmacologically defined binding site, the TXA₂/PGH₂ receptor.³ The half-life periods of TXA₂ and PGH₂ in aqueous solution at physiological pH are 30 seconds and 5 minutes, respectively.⁴ To date, several thromboxane analogues have been synthesized that are stable in aqueous solutions. In this context, it has been demonstrated that the TXA₂ mimetic 9,11-epithio-11,12-methano-TXA₂ enhances DNA synthesis in VSMCs.⁵ It has been suggested that TXA₂ generation may also occur locally within the vascular wall and that this may contribute in part to media hypertrophy observed in hypertension. It is generally accepted that the endothelium produces a number of vasoconstrictors and growth factors, such as TXA₂ and PDGF.^{6 7} PDGF is a potent proliferative factor in vitro for different cell species including VSMCs, thus it is postulated that it plays an important role in the pathogenesis of atherosclerosis.⁸

To elucidate possible intracellular pathways for the transmission of growth signals by TXA₂, we investigated the effects of the TXA₂ mimetics CTA₂⁹ and U46619 [(15S)-hydroxy-11,9-(epoxymethano)prosta-5Z,13E-dienoic acid], a stable PGH₂ analogue acting through the common TXA₂/PGH₂ receptor,³ on [Ca²⁺]_i, MAP kinase activity, and egr-1 and c-fos expression. In addition, we examined the effect of different concentrations of CTA₂ and U46619 alone and in combination with PDGF-BB on cell DNA synthesis and cell number. To examine whether the effect of the TXA₂ mimetics on cell growth is mediated by PTX-sensitive G_i protein, we investigated the influence of PTX on TXA₂ mimetic–induced DNA synthesis.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Isolation of VSMCs
Rat VSMCs were isolated by enzymatic dispersion with modification of the method described by Chamley et al.¹⁰ The thoracic aortas from Wistar-Kyoto rats (6 to 8 weeks old, Charles River Wiga GmbH) were removed and transferred on ice in PBS containing 1% penicillin/streptomycin (wt/vol). The aorta was freed from connective tissue; was transferred into a Petri dish containing 3 mL of an enzyme dissociation mixture containing DMEM with 400 IU/mL collagenase type I, 0.5 mg/mL elastase, and 0.5 mg/mL soybean trypsin inhibitor; and was incubated for 30 minutes at 37°C. Then the aorta was transferred into Dulbecco’s PBS, and the adventitia was stripped off with forceps under a binocular microscope. The aorta was minced, and the minced media was transferred into a plastic tube containing 6 mL of the enzyme dissociation mixture. The suspension was incubated for up to 2 hours at 37°C under constant agitation and then centrifuged (1500 rpm for 10 minutes). The pellet was resuspended in DMEM with 10% fetal calf serum and plated in a Petri dish (diameter, 3 cm). Cells were cultured over several passages according to Ross.¹¹ VSMCs were cultured in DMEM supplemented with 10% fetal calf serum, nonessential amino acids, 100 IU/mL penicillin, and 100 µg/mL streptomycin in 75-cm² flasks at 37°C in a humidified atmosphere of 95% air and 5% CO₂ (Steri-cult incubator, Forma Scientific). VSMCs in culture showed the characteristic hill-and-valley growth pattern. The purity of VSMCs was confirmed by immunocytochemical localization of smooth muscle–specific -smooth muscle actin with the use of fluoresceinisothiocyanate–conjugated monoclonal anti–-smooth muscle actin plus a fluorescein isothiocyanate-conjugated F(ab')2 fragment of goat anti-mouse immunoglobulin. Experiments were performed with three different cell lines in passages 6 to 15.

Measurement of [Ca²⁺]_i
For measurement of [Ca²⁺]_i, confluent cells were detached with 0.04% trypsin/0.02% EDTA in PBS after 5 to 10 minutes at 37°C. The cells were then cultured on round glass microscope slides (diameter, 12 mm) under normal tissue culture conditions. When the cells became confluent they were incubated with 2 µmol/L fura 2-AM at 37°C for 20 minutes in HEPES buffer (20 mmol/L HEPES, 16 mmol/L glucose, 130 mmol/L NaCl, 1 mmol/L MgSO₄ · 7H₂O, 0.5 mmol/L CaCl₂, Tris-base, pH 7.4) supplemented with 1% bovine serum albumin (wt/vol). Just before measurements the cell monolayer was rinsed with HEPES buffer containing 1 mmol/L CaCl₂, and the glass slide was positioned diagonally in the cuvette. Ca²⁺–fura 2 fluorescence was measured at 37°C in a fluorescence spectrofluorometer (Hitachi) at excitation wavelengths of 340 and 380 nm and an emission wavelength of 505 nm. Maximum (R_max) and minimum (R_min) fluorescence was determined by addition of digitonin at a final concentration of 3x10^-5 mol/L followed by the addition of Tris-base/EGTA at a final concentration of 0.1 mol/L Tris-base and 25 mmol/L EGTA. Fluorescence was corrected for cellular autofluorescence. Fluorescence signals were calibrated according to Grynkiewicz et al¹² with the following equation: [Ca²⁺]_i=K_dx(R-R_min)/(R_max-R)x(S_f2/S_b2). K_d for the fura 2–Ca²⁺ complex at 37°C is assumed to be 224 nmol/L.¹² S_f2 is the 380 nm–excited fluorescence in the absence of Ca²⁺ (EGTA added), and S_b2 is the 380 nm–excited fluorescence in the presence of saturating Ca²⁺ (1 mmol/L Ca²⁺).

Immunoblotting
MAP kinase protein analysis was performed by a modification of the enhanced chemiluminescence Western blotting method as previously described by Simm et al.¹³ VSMCs were seeded in 24-well culture plates (4x10⁵ cells per well; well diameter, 12 mm) and cultivated in culture medium until confluent. Then the medium was replaced by serum-free (quiescent) medium consisting of a mixture of DMEM and Ham’s F-10 medium (1:1). After another 24 hours of cultivation in quiescent medium, cells were stimulated for different time periods. The medium was removed, and cells were lysed with a buffer containing 50 mmol/L Tris-HCl (pH 6.7), 2% SDS, 2% mercaptoethanol, and 1 mmol/L sodium orthovanadate. Samples were vigorously shaken for 5 minutes, and 2.5 µL benzonase (250 U/µL) was added to digest nucleic acids. After 5 minutes of vigorous shaking, sample solutions were transferred into microtest tubes and 6 µL bromphenol blue in 50% glycerol was added. Aliquots were used for protein determinations with the use of the Bio-Rad protein assay according to the method of Bradford.¹⁴ Thirty micrograms of protein was analyzed by SDS-PAGE in a 7.5% acrylamide gel (thickness, 0.75 mm) with the use of the Mini Gel Protean system (Bio-Rad). The prestained standards from Bio-Rad were used as molecular weight standards consisting of myosin (204 000), ß-galactosidase (132 000), bovine serum albumin (65 000), carbonic anhydrase (42 000), soybean trypsin inhibitor (29 000), lysozyme (17 100), and aprotinin (6500). Proteins were transferred to a polyvinylidene difluoride membrane overnight by 100 mA with a buffer containing 25 mmol/L Tris-base, 192 mmol/L glycine, and 20% methanol, pH 8.3. The protein transfer was checked with Ponseau S. The membrane was washed three times with 50 mmol/L Tris-Cl and 150 mmol/L NaCl, pH 7.5. Saturation was performed with 50 mmol/L Tris-Cl and 150 mmol/L NaCl, pH 7.5, containing 2% bovine serum albumin (wt/wt), 0.03% NaN₃, and 0.2% Nonidet P-40 (saturation buffer). The sheets were incubated for 1 hour with saturation buffer without NaN₃ containing 5 µg/mL anti-rat MAP kinase R2 IgG for detection of MAP kinases. After six quick rinses with washing buffer containing 0.5% bovine serum albumin and 0.2% Nonidet P-40 without NaN₃ (washing buffer), sheets were incubated for 1 hour in saturation buffer containing horseradish peroxidase–labeled donkey anti-rabbit immunoglobulin G (1:5000 dilution). Antibodies were removed after being washed six times with washing buffer without NaN₃. The detection of proteins was performed using the chemiluminescence method with luminol. Sheets were soaked in 3 mL chemiluminescence buffer containing 100 mmol/L Tris-Cl (pH 8.5), 2.5 mmol/L luminol, and 400 µmol/L p-coumaric acid, to which 3 mL hydrogen peroxide buffer was added (5.4 mmol/L H₂O₂ and 100 mmol/L Tris-Cl, pH 8.5). After 1 minute sheets were dried with filter papers and wrapped in transparent foil. Light emission was detected with films (Kodak X-OMAT, 8x10 in). Stock solutions of luminol (250 mmol/L) and p-coumaric acid (90 mmol/L) in dimethyl sulfoxide were used.

RNA Extraction and Analysis
The expression of egr-1 and c-fos mRNA was studied after preincubation of cells for 24 hours in serum-free quiescent medium in 75-cm² culture flasks. Then the quiescent cells were stimulated with CTA₂ or U46619 for different time periods. Total RNA was extracted from VSMCs by the guanidinium isothiocyanate/CsCl procedure.¹⁵ Typically, between 50 and 70 µg total RNA was obtained from the cells of a 75-cm² flask. Northern blotting was performed as previously described.¹⁶ Ten micrograms total RNA was separated by electrophoresis in a 6% formaldehyde/1.2% agarose gel, blotted on Hybond N⁺ membranes (Amersham), washed at room temperature in 5x SSC (1x SSC is 0.15 mol/L NaCl, 0.015 mol/L sodium citrate) for 5 minutes, and fixed with UV irradiation. After fixing, the blots were washed at 60°C in 0.1x SSC and 0.1% SDS for 5 minutes. Prehybridization and hybridization were performed overnight at 60°C in 5x SSC, 0.2% SDS, 50 mmol/L sodium phosphate, 10x Denhardt’s solution (Sigma Chemical Co), and 200 µg/mL salmon sperm DNA. The DNA probes were labeled with [³²P]dCTP by random oligonucleotide priming to a specific activity of 2 to 4x10⁹ disintegrations per minute/µg DNA (Amersham Buchler). The stringency of the final wash was 0.2x SSC containing 0.1% SDS at 65°C for 2x45 minutes. A 2.1-kb fragment (OC68 insert) of egr-1 including three zinc-finger domains was used as a probe.¹⁷ Blots were exposed to films (Kodak X-OMAT, 8x10 in) for 3 to 7 days at -70°C. Blots were standardized with a 0.77-kb cDNA probe for ß-actin (Dianova/Oncor Science). The size in kilobases of the detected mRNA was calculated by the 18S (1.8-kb) and 28S (4.6-kb) ribosomal RNA migration from the gel wells.

Determination of DNA Synthesis and Cell Number
The effects of PDGF-BB, CTA₂, and U46619 on DNA synthesis were measured by a slight modification of methods described previously.¹⁸ VSMCs were seeded in 24-well culture plates and cultivated in culture medium until confluent. Then the medium was replaced by serum-free (quiescent) medium consisting of a mixture of DMEM and Ham’s F-10 medium (1:1). After another 24 hours of cultivation in quiescent medium, stimulators were added to the cells. Cultures were exposed to the stimulating agents for 20 hours before 3 µCi/mL [³H]thymidine was added to the quiescent medium. Four hours later experiments were terminated by aspirating the medium and subjecting the cultures to sequential washes with PBS containing 1 mmol/L CaCl₂, 1 mmol/L MgCl₂, 10% trichloroacetic acid, and ethanol/ether (2:1, vol/vol). Phase-contrast microscopy was used to inspect the dishes for evidence of cell detachment or changes in cell morphology. Acid-insoluble [³H]thymidine was extracted into 250 µL per dish of 0.5 mol/L NaOH, and 0.1 mL of this solution was mixed with 5 mL scintillator (Packard, Ultima Gold) and quantified with the use of a liquid scintillation counter (model Beckmann, LS 3801). Fifty microliters of the residual solution was prepared for the determination of protein with the Bio-Rad protein assay according to the method of Bradford.¹⁴

For cell counting, VSMCs were seeded in 24-well culture plates (5x10⁴ cells per well; well diameter, 12 mm) and cultured in DMEM supplemented with 10% fetal calf serum, nonessential amino acids, 100 IU/mL penicillin, and 100 µg/mL streptomycin at 37°C for 24 hours. Under these conditions cell confluence of approximately 70% was reached. The medium was then replaced by serum-free quiescent medium consisting of DMEM and Ham’s F-10 (1:1, vol/vol), and VSMCs were stimulated with CTA₂ and U46619 in the presence and absence of PDGF-BB. After 24 hours cells were trypsinized and resuspended in DMEM plus trypan blue. Cell counting was performed with a Neubauer cell-box by light microscopy.

Determination of PTX-Induced [³²P]ADP Ribosylation
PTX-induced [³²P]ADP ribosylation was determined according to Böhm et al¹⁹ with minor modifications. VSMCs were seeded in 75-cm² culture flasks and cultivated in culture medium until confluent. Medium was replaced by quiescent medium consisting of a mixture of DMEM and Ham’s F-10 medium (1:1), and then cells were preincubated with different PTX concentrations for 24 hours. Cells were washed three times with ice-cold PBS buffer and scraped with a rubber policeman. After cells were centrifuged at 3000g for 15 minutes, pellets were homogenized with ice-cold homogenization buffer consisting of 5 mmol/L Tris-Cl, 1 mmol/L EDTA, 5 mmol/L MgCl₂, 1 mmol/L dithiothreitol, 0.5 µg/mL leupeptin, and 0.5 µg/mL aprotinin, pH 7.4, incubated at 4°C for 15 minutes, and homogenized for 1 minute with a glass-polytetrafluoroethylene homogenizer. The homogenate was spun at 484g (Beckman JA 20 rotor) for 10 minutes. The supernatant was centrifuged at 48 000g for 20 minutes and discarded, and the pellet was resuspended in 1 mL of a solution consisting of 25 mmol/L Tris-Cl, 1 mmol/L EGTA, and 1 mmol/L dithiothreitol, pH 8.0. Protein determination was performed according to Bradford.¹⁴

Membranes from control and PTX-treated VSMCs were [³²P]ADP ribosylated in vitro to demonstrate whether complete inhibition of the PTX-sensitive G_i proteins occurred after PTX treatment. [³²P]ADP ribosylation was performed as described previously.¹⁹ Briefly, membranes (0.5 µg) were incubated at 4°C for 18 hours in 50 µL of a medium containing 100 mmol/L Tris-HCl (pH 8.0), 25 mmol/L dithiothreitol, 2 mmol/L ATP, 1 mmol/L GTP, 0.1% (vol/vol) Lubrol, 50 nmol/L ³²P-NAD (800 Ci/mmol), and 20 µg/mL PTX that had been activated by incubation with 50 mmol/L dithiothreitol for 1 hour at 20°C before the labeling reaction. Samples were subjected to SDS-PAGE in a 10% acrylamide gel. Gels were stained with Coomassie blue and dried before autoradiography was performed.

Materials
CTA₂, TXB₂, and PTX were obtained from Calbiochem. U46619 was a gift from Upjohn Co. SQ29548 ([1S]-1,2ß(5Z),3ß,4-7-(3-{2-[(phenylamino)carbonyl]hydrazino}methyl)-7-oxabicyclo[2.2.1]hept-2-yl-5-heptanoic acid) was a gift from Dr M. Ogletree (Squibb Institute for Research, Princeton, NJ). Anti-rat MAP kinase R2 IgG was purchased from Paesel and Lorei. Anti–-smooth muscle actin was obtained from Sigma Chemical Co. Fluorosceinisothiocyanate-conjugated F(ab')2 fragment of goat anti-mouse immunoglobulins was obtained from Dako GmbH. DMEM, Ham’s F-10, and Dulbecco’s PBS were obtained from Gibco BRL. A 2.1-kb fragment (OC68 insert) of egr-1 including three zinc-finger domains was used as a DNA probe.¹⁷ A 0.77-kb cDNA for ß-actin (Dianova/Oncor Science) was used as a DNA probe for ß-actin. Hybond N⁺ membranes, [³²P]dCTP, and [methyl-³H]thymidine were obtained from Amersham. X-OMAT 8x10-in films were obtained from Eastman Kodak. Molecular weight standard RNA was obtained from Boehringer Mannheim. Polyvinylidene difluoride membranes were obtained from Millipore.

Statistics
Values are expressed as mean±SEM unless noted as mean±SD. Statistical analysis of the data was performed with the Mann-Whitney U test (STATVIEW 512⁺, version 1.0, Apple Computer Inc). Triplicate wells were analyzed for each [³H]thymidine incorporation experiment, and each experiment was performed independently a minimum of three times. Data presented are from representative experiments unless otherwise indicated. Data obtained from individual experiments with triplicate determinations were normalized by calculation of the mean±SEM of the individual experiments and are expressed as percent change from the basal value of unstimulated cells (=100%). A value of P<.05 was considered statistically significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Effect of CTA₂ on [Ca²⁺]_i
As demonstrated in Fig 1A, 10^-6 mol/L CTA₂ in the presence of extracellular Ca²⁺ induced a maximal elevation in [Ca²⁺]_i from approximately 100 to 300 nmol/L at 25 seconds (a, b, and c represent tracings from one experiment). [Ca²⁺]_i declined after the peak toward a stable [Ca²⁺]_i value of approximately 220 nmol/L within 3 minutes. Evaluation of three separate experiments was performed by calculation of the maximal increase in [Ca²⁺]_i at 25 seconds. CTA₂ at 10^-6 mol/L caused an increase in [Ca²⁺]_i from 88±10 (basal value) to 270±30 nmol/L (mean±SD, n=3, P<.05) at 25 seconds. In the absence of extracellular Ca²⁺, CTA₂ induced an increase in [Ca²⁺]_i from 60 to 180 nmol/L, with a maximum at 30 seconds (Fig 1b). [Ca²⁺]_i declined after this peak value toward basal values within 4 minutes. Evaluation of three independent experiments revealed that CTA₂ in the absence of extracellular Ca²⁺ induced an increase in [Ca²⁺]_i from 55±10 to 165±40 nmol/L (mean±SD, n=3, P<.05) at 30 seconds. In contrast, 10^-6 mol/L TXB₂ did not influence [Ca²⁺]_i in the presence of extracellular Ca²⁺ (Fig 1c). Subsequent addition of Ang II (10^-7 mol/L) induced a marked increase in [Ca²⁺]_i to 400 nmol/L, with a maximum at 20 seconds. CTA₂ at 10^-9 to 10^-7 mol/L lacked any effect on [Ca²⁺]_i. As shown in Fig 1B, treatment of cells with PTX (100 ng/mL) for 24 hours did not affect the CTA₂ and U46619 effects on [Ca²⁺]_i, whereas treatment of cells with SQ29548 (10^-6 mol/L) for 3 minutes caused an almost 100% inhibition of the TXA₂ mimetic–induced [Ca²⁺]_i increase. PTX or SQ29548 per se had no effects on basal [Ca²⁺]_i.

View larger version (16K): [in this window] [in a new window]   Figure 1. Effect of CTA2 on [Ca2+]i in cultured rat VSMC monolayers. A, CTA2 (10-6 mol/L), TXB2 (10-6 mol/L), and Ang II (10-7 mol/L) were applied to fura 2–loaded VSMC monolayers, and changes in fluorescence were monitored. After subtraction of autofluorescence, changes in the 340/380 nm excitation wavelength ratio by the emission wavelength of 505 nm were converted into corresponding [Ca2+]i levels. a, Effect of CTA2 (10-6 mol/L) on [Ca2+]i in the presence of extracellular Ca2+. b, Effect of CTA2 (10-6 mol/L) on [Ca2+]i in the absence of extracellular Ca2+ (Ca2+-free HEPES buffer containing 1 mmol/L EGTA). c, Effect of TXB2 (10-6 mol/L) and Ang II (10-7 mol/L) on [Ca2+]i in the presence of extracellular Ca2+. B, Effect of PTX (100 ng/mL) or SQ29548 (10-6 mol/L) on TXA2 mimetics effect on [Ca2+]i. Evaluation of three separate experiments was performed by calculating percent inhibition of TXA2 mimetics effect on maximal increase in [Ca2+]i at 25 seconds. Cells were stimulated with TXA2 mimetics (10-6 mol/L) after preincubation of cells with SQ29548 (10-6 mol/L) for 3 minutes. PTX experiments were performed as follows: Cells were cultured on round glass microscope slides (diameter, 12 mm) under normal tissue culture conditions. When cells became confluent, medium was replaced by serum-free (quiescent) medium consisting of a mixture of DMEM and Ham’s F-10 medium (1:1) in the presence and absence of 100 ng/mL PTX for 24 hours. Then cells were loaded with fura 2-AM, and [Ca2+]i was measured.

Effect of CTA₂ and U46619 on the Phosphorylation of MAP Kinase
MAP kinases, in particular the MAP-I isoform migrating with an apparent molecular weight of 44 kD (p44^mapk) and the MAP-II isoform migrating with a molecular weight of 42 kD (p42^mapk), are activated by threonine and tyrosine phosphorylation in response to different growth factors.²⁰ The applied anti–MAP kinase antibodies are able to recognize both MAP kinase isoforms. The activity of the 42-kD MAP-II kinase isoform can be monitored by the electrophoretic mobility of the phosphorylated MAP-II kinase, which possesses a higher apparent molecular weight compared with the inactivated form, resulting in a decrease in the electrophoretic mobility of the phosphorylated MAP-II kinase. Fig 2 shows the time course for the shift in mobility of the 42-kD MAP-II kinase band after cell stimulation with PDGF-BB (20 ng/mL), CTA₂, and U46619 (both 10^-6 mol/L). When cells were stimulated with PDGF-BB, the 42-kD band was maximally shifted (100%) at 3 to 5 minutes (no detectable amount of the unstimulated 42-kD MAP-II kinase was observed). Approximately 60% was shifted at 1 minute (intensity of the 42-kD band plus intensity of the shifted 42-kD band=100%), approximately 80% at 10 minutes, and approximately 20% at 15 minutes. When cells were stimulated with CTA₂ or U46619, approximately 20% of the 42-kD band was shifted at 1 minute. Fifty percent of the 42-kD band was shifted at 5 to 10 minutes. After 15 minutes no shift could be observed, demonstrating that the 42-kD MAP-II kinase was no longer phosphorylated. Parallel cell stimulation with PDGF-BB for 10 minutes resulted in an approximate 50% shift of the 42-kD band in these experiments.

View larger version (27K): [in this window] [in a new window]   Figure 2. Effect of CTA2 and U46619 on 42-kD MAP kinase activity. VSMCs were seeded in 24-well culture plates (4x105 cells per well; well diameter, 12 mm) and cultivated in culture medium until confluent. Then the medium was replaced by serum-free medium. After another 24 hours of cultivation in quiescent medium, cells were stimulated with CTA2 (10-6 mol/L), U46619 (10-6 mol/L), and PDGF for different time periods. Cells were lysed in buffer containing SDS. Thirty micrograms protein was analyzed by SDS-PAGE. MAP kinase was detected after blotting on polyvinylidene difluoride membranes by a specific MAP kinase antibody that recognizes the two MAP kinase isoenzymes.

Effect of CTA₂ and U46619 on c-fos and egr-1 mRNA Expression
Fig 3 shows the time course of the 3.4-kb egr-1 and 2.2-kb c-fos mRNA expression induced after stimulation of VSMCs with CTA₂ or U46619 (each 10^-6 mol/L). CTA₂ and U46619 induced a maximal induction of egr-1 and c-fos mRNA at 30 minutes. After 60 minutes the values returned to control levels.

View larger version (60K): [in this window] [in a new window]   Figure 3. Effects of CTA2 on the c-fos (A) and egr-1 (B) mRNA levels and effects of U46619 on the c-fos (C) and egr-1 (D) mRNA levels in VSMC. Confluent cells in 75-cm2 culture flasks were preincubated in serum-free quiescent medium for 24 hours, and then cells were stimulated with CTA2 (10-6 mol/L) or with U46619 (10-6 mol/L). Total RNA was extracted by the guanidinium isothiocyanate/CsCl procedure. Ten micrograms of total RNA was separated on a formaldehyde-agarose gel, blotted on Hybond N+ membranes, and probed with a 32P-labeled 2.1-kb fragment of egr-1, which hybridized to the 3.4-kb egr-1 mRNA, or with a 32P-labeled 1.0-kb v-fos cDNA, which hybridized to the 2.2-kb mRNA of c-fos. The same membranes were rehybridized with a 0.77-kb cDNA probe for ß-actin. Arrows indicate 28S (4.6-kb) and 18S (1.8-kb) ribosomal RNA.

Effect of CTA₂ and U46619 Alone or in Combination With PDGF-BB on Cell DNA Synthesis
The effect of different concentrations of CTA₂ alone and in combination with PDGF-BB (20 ng/mL) on cell DNA synthesis is shown in Fig 4A (one representative experiment performed in triplicate wells). CTA₂ alone at final concentrations of 10^-9 to 10^-7 mol/L did not influence either basal or PDGF-BB–dependent cell DNA synthesis. PDGF-BB (20 ng/mL) and CTA₂ (10^-6 mol/L) induced increases in [³H]thymidine incorporation into cell DNA from 124±10 to 913±183 and 608±93 cpm/µg protein (mean±SD of the triplicate determination), respectively. Exposure of rat VSMCs to both PDGF-BB (20 ng/mL) and CTA₂ (10^-6 mol/L) enhanced PDGF-BB–dependent [³H]thymidine incorporation from 913±183 to 2114±175 cpm/µg protein. Data from individual experiments each performed with triplicate wells were normalized by calculation of the mean±SEM of the individual experiments and were expressed as percent increase above the basal value of unstimulated cells (=100%) (Fig 4B). These results show that 24 hours after cell treatment PDGF-BB (20 ng/mL) and CTA₂ (10^-6 mol/L) caused a 552±86% (n=6, P<.05) and 334±42% (n=6, P<.05) increase in [³H]thymidine incorporation above basal values, respectively. Exposure of rat VSMCs to both PDGF-BB (20 ng/mL) and CTA₂ (10^-6 mol/L) resulted in a 1936±438% increase over basal values (n=3, P<.05). These results show that CTA₂ caused a 3.5-fold elevation of the PDGF-BB–dependent effect on [³H]thymidine incorporation (P<.05 for the PDGF-BB effect versus the effect of PDGF-BB plus CTA₂). U46619 (10^-6 mol/L) induced a 183±27% increase in [³H]thymidine incorporation over basal values (n=4, P<.05), demonstrating that U46619 at a similar concentration is less efficient than CTA₂. Treatment of cells with 10^-5 mol/L U46619 resulted in a 713±138% increase in [³H]thymidine incorporation above the basal value (n=4, P<.05). Cotreatment of cells with PDGF-BB (20 ng/mL) and U46619 (10^-6 and 10^-5 mol/L) resulted in 918±128% and 1357±64% increases over basal values (n=3, P<.05), respectively, demonstrating a 1.7- and 2.5-fold elevation of the PDGF-BB–dependent effect (P<.05 for the PDGF-BB effect per se versus the effect of PDGF-BB in the presence and absence of 10^-6 and 10^-5 mol/L U46619). These results suggest that CTA₂ and U46619 at 10^-6 mol/L exerted a synergistic effect on the PDGF-BB–dependent effect on cell DNA synthesis, whereas U46619 at 10^-5 mol/L possessed a rather additive effect. To show that the growth-promoting effects of CTA₂ and U46619 on VSMC DNA synthesis are mediated by TXA₂/PGH₂ receptors, we investigated the effect of SQ29548 on CTA₂- and U46619-dependent cell DNA synthesis. SQ29548 is known to be a selective TXA₂/PGH₂ receptor antagonist.²¹ At 10^-6 mol/L, SQ29548 completely blunted the effect of both agonists (10^-6 mol/L) down to basal values and induced an approximately 40% reduction of the U46619 effect at 10^-5 mol/L (P<.05 for CTA₂ or U46619 in the presence of SQ29548 versus CTA₂ or U46619).

View larger version (23K): [in this window] [in a new window]   Figure 4. Effect of PGH2/TXA2 mimetics on DNA synthesis in VSMCs. A, Confluent cells (24-well plates) were precultured for 24 hours in quiescent medium. CTA2 at concentrations of 10-9, 10-8, 10-7, or 10-6 mol/L was added 5 minutes before stimulation with 20 ng/mL PDGF-BB. After another 20-hour incubation, cells were exposed to 3 µCi/mL [3H]thymidine. Four hours later the reaction was terminated, and cell protein and [3H]thymidine incorporation into cell DNA were quantified. Panel A represents one representative experiment performed in triplicate wells. Values are mean±SD. B, Effect of U46619 and CTA2 in the presence and absence of PDGF-BB or SQ29548. Data from individual experiments each performed with triplicate wells were standardized by calculating the mean±SEM of individual experiments and are expressed as percent increase above the basal value of unstimulated cells (=100%). SQ29548 (10-6 mol/L) was added 5 minutes before cell stimulation. *P<.05 for PDGF-BB or CTA2 vs control, n=6; *P<.05 for PDGF-BB+CTA2 vs control, n=3; *P<.05 for 10-6 or 10-5 mol/L U46619 vs control, n=4; **P<.05 for PDGF-BB+CTA2 vs PDGF-BB, n=3; **P<.05 for PDGF-BB+U46619 vs control, n=3; ***P<.05 for U46619 or CTA2 in the presence of SQ29548 vs U46619 or CTA2, n=3.

Effect of CTA₂ and U46619 Alone or in Combination With PDGF-BB on Cell Number
Fig 5 shows the effects of CTA₂ and U46619 (10^-6 mol/L) per se and in combination with PDGF-BB on cell number. CTA₂, U46619, and PDGF-BB per se all induced an increase in cell number from 4.31x10⁵±5.3x10⁴ cells per milliliter (basal value) to 6.19x10⁵±3.06x10⁴, 6.65x10⁵±9.28x10⁴, and 8.26x10⁵±5.30x10⁴, respectively (mean±SD, P<.05). CTA₂ and U46619 in combination with PDGF-BB enhanced the PDGF-BB–dependent increase in cell counts from 8.26x10⁵ to 1.09x10⁶±2.65x10⁴ and to 1.19x10⁶±3.97x10⁴ cells per milliliter, respectively (mean±SD, n=3, P<.05).

View larger version (64K): [in this window] [in a new window]   Figure 5. Effects of TXA2/PGH2 mimetics on VSMC cell number. Cells (5x104) were seeded per well (24-well plates) in cultured medium containing 10% fetal calf serum. After 24 hours approximately 70% confluence was reached. Then the medium was replaced with quiescent medium, and cells were immediately stimulated with CTA2 and U46619 (10-6 mol/L) in the presence and absence of PDGF-BB (20 ng/mL). After 24 hours cells were trypsinized and counted as described in "Methods." Data represent mean±SD of a representative experiment performed in triplicate. *P<.05 vs control; **P<.05 for PDGF-BB vs PDGF-BB+U46619 or CTA2.

Effect of PTX on CTA₂- and U46619-Dependent Cell DNA Synthesis
To elucidate whether PTX-sensitive G_i proteins are involved in TXA₂/PGH₂ mimetic–induced VSMC growth, we tested the effect of PTX on CTA₂- and U46619-dependent [³H]thymidine incorporation. As demonstrated in Fig 6, pretreatment of cells with 10 or 100 ng/mL PTX for 24 hours had no effect on CTA₂- and U46619-induced [³H]thymidine incorporation, suggesting that G_i proteins are not involved in the TXA₂ mimetic–induced VSMC growth.

View larger version (48K): [in this window] [in a new window]   Figure 6. Effect of PTX on PGH2/TXA2 mimetic–induced DNA synthesis in VSMCs. Confluent cells (24-well plates) were precultured for 24 hours in the quiescent medium in the presence and absence of PTX. Then CTA2 (10-6 mol/L), U46619 (10-5 mol/L), or PDGF-BB (20 ng/mL) was added to cells. After another 20 hours of incubation, cells were exposed to 3 µCi/mL [3H]thymidine. Four hours later the reaction was terminated, and cell protein and [3H]thymidine incorporation into cell DNA were quantified. Values are mean±SD.

Effect of PTX Treatment of VSMCs on [³²P]ADP Ribosylation of PTX-Sensitive G Proteins
To establish whether pretreatment of VSMCs with PTX for 24 hours leads to a complete inactivation of the PTX-sensitive G proteins (G_i, G_t, and G_o), we investigated the [³²P]ADP ribosylation of PTX-sensitive G proteins in PTX-treated and -untreated cells. The entire disappearance of a protein of an apparent molecular weight (M_r) of 39 to 41 kD in the PTX-treated cells would suggest a complete inactivation of the PTX-sensitive G_i proteins. As expected, incubation of the non–PTX-treated VSMC membranes with PTX and ³²P-NAD led to a remarkable ADP ribosylation of the -subunit of the PTX-sensitive G proteins, as indicated in Fig 7. The PTX-sensitive G_t protein transducin (M_r=39 kD) was used as a positive control. [³²P]ADP ribosylation revealed a protein band with an apparent M_r of 39 to 41 kD. Pretreatment of VSMCs with 10 or 100 ng/mL PTX for 24 hours entirely blocked the PTX-induced ADP ribosylation of the PTX-sensitive -subunit of the G protein. Pretreatment of the cells with 1 ng/mL led to a 90% inhibition of the ADP ribosylation (as determined by densitometry).

View larger version (63K): [in this window] [in a new window]   Figure 7. Effect of PTX treatment of VSMCs on ADP ribosylation of PTX-sensitive G proteins. Confluent cells were precultured in quiescent medium in the presence and absence of PTX for 24 hours. Cell membranes from PTX-treated and -untreated cells were prepared as described in "Methods." Membranes (0.5 µg) were incubated with 32P-NAD in ribosylation medium at 4°C for 18 hours in the presence of activated PTX (20 µg/mL). Samples were subjected to SDS-PAGE in a 10% acrylamide gel. Gels were stained with Coomassie blue and dried before autoradiography. Transducin (100 ng) was used as a positive control.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The eicosanoids PGH₂ and TXA₂ produced by stimulated platelets and vessel tissues exert potent vasoconstricting effects on VSMCs that are mediated by the common TXA₂/PGH₂ receptor.^{3 4} The present study shows that the TXA₂/PGH₂ mimetics CTA₂ and U46619 at 10^-6 mol/L induce an elevation in [Ca²⁺]_i, activate the 42-kD MAP kinase, stimulate the expression of immediate egr genes, and promote proliferation of VSMCs. In contrast, TXB₂, which is formed after hydrolysis of TXA₂, lacked any significant effects on [Ca²⁺]_i and DNA synthesis in VSMCs. Furthermore, we demonstrated that the growth-promoting effects of the TXA₂ mimetics are insensitive to PTX, suggesting that the TXA₂/PGH₂ receptor is not coupled to a G_i protein.

G proteins are heterotrimers consisting of three distinct subunits: (39 to 46 kD), ß (37 kD), and (8 kD) (for review see Reference 22²² ). It is assumed that in the basal state G proteins exist in the G_ß form with tightly bound GDP. After interaction of a hormone with its receptor, GDP is replaced by GTP, which binds to the -subunit, creating the active form of the G proteins that is associated with the receptor-hormone complex. After G_ß-subunit and subsequent receptor-hormone dissociation, the G-subunit–GTP complex mediates the activity of downstream effector proteins such as adenylate cyclase, phospholipase C, and phospholipase A. Inactivation of the G-subunit–GTP complex occurs by the intrinsic GTPase activity of the G-subunit. So far, 21 G protein -subunits have been identified. These are divided into four major subfamilies: G_s, G_i, G_q, and G₁₂. The intrinsic GTPase activity of the G_i subfamily can be inhibited by PTX via ADP ribosylation of specific residues. Recent reports demonstrate that the PTX-insensitive G_q subfamily can couple to several receptors and thereby modulate the specific phospholipase C_ß1, which catalyzes the hydrolysis of phosphatidylinositol bisphosphate, with subsequent formation of IP₃ and diacylglycerol and an increase in [Ca²⁺]_i.^{23 24}

Using PTX (10 to 100 ng/mL) we demonstrated that G_i proteins are not involved in TXA₂ mimetic–induced VSMC growth. In accordance with this finding we also observed that treatment of cells with 100 ng/mL PTX for 24 hours did not alter the CTA₂- and U46619-induced increase in [Ca²⁺]_i. Recently, it has been demonstrated that the TXA₂/PGH₂ receptor–coupled G protein in human platelets belongs to a member of the G_q subfamily, possesses an apparent molecular weight of 42 kD, and mediates the TXA₂ stimulation of phospholipase C, with subsequent formation of IP₃ and diacylglycerol and an increase in [Ca²⁺]_i.^{25 26}

It has been demonstrated that stimulation of VSMCs with the TXA₂ mimetic U46619 resulted in an increase of IP₃, [Ca²⁺]_i, and c-fos mRNA.²⁷ In accordance with the results of Dorn et al,²⁷ we have demonstrated that CTA₂, another TXA₂ mimetic, increases [Ca²⁺]_i. This increase was in part maintained in the absence of extracellular Ca²⁺, suggesting that CTA₂ is able to mobilize Ca²⁺ from internal stores. We also observed that 10^-6 mol/L U46619 induced an increase in [Ca²⁺]_i that was approximately 60% of that induced by 10^-6 mol/L CTA₂ (data not shown).

As demonstrated by Dorn et al,²⁷ U46619 induces a phospholipase C–dependent increase in IP₃. From these findings it can be concluded that the intracellular signaling in VSMCs by TXA₂ mimetics is mediated by G_q proteins. Similarly to Dorn et al we were able to demonstrate that CTA₂ mobilized Ca²⁺ from intracellular stores, suggesting a phospholipase C/IP₃-dependent mechanism and consequently a G_q protein–mediated effect. Treatment of cells with PTX (100 ng/mL) for 24 hours did not influence the effect of CTA₂ and U46619 on cell DNA synthesis. In accordance with these findings we observed that treatment of cells with PTX for 24 hours did not affect CTA₂ and U46619 effects on [Ca²⁺]_i. Entire inactivation of the PTX-sensitive G_i proteins was shown by [³²P]ADP ribosylation of isolated VSMC membranes from PTX-treated and untreated cells. These findings suggest that G_i proteins are not involved in the TXA₂ growth-promoting effects in VSMCs. Furthermore, we can conclude that as in platelets, the TXA₂/PGH₂ receptor–coupled G protein belongs to the G_q subfamily.

In general, it has been established that a number of vasoconstrictive peptides, such as Ang II^{28 29 30 31} and thrombin,^{32 33} are potent growth factors for VSMCs acting via binding to their specific receptors that are coupled to G proteins. On binding to their G protein–coupled specific receptors on VSMCs, Ang II³⁴ and thrombin³⁵ activate phospholipase C, resulting in the accumulation of diacylglycerol and IP₃ and leading to mobilization of Ca²⁺ from intracellular stores and the stimulation of protein kinase C. [Ca²⁺]_i and stimulation of protein kinase C are important intracellular signaling mechanisms by which growth factors regulate many different processes, such as contraction and cell growth.³⁶

It is well known that Ang II is also capable of stimulating VSMCs^{28 29 30 31} via the type 1 receptor, which belongs to the G_q protein–coupled receptors.^{37 38} In this context, it has been demonstrated that the Ang II–induced stimulation of the phosphatidylinositol turnover signaling system in VSMCs is not inhibited by PTX.³⁹ However, VSMC³³ and Chinese hamster lung fibroblast⁴⁰ treatment with PTX (1 to 10 ng/mL) resulted in a strong attenuation of thrombin-induced DNA synthesis in both cell types, suggesting an implication of G_i proteins in thrombin-induced cell growth. It has been reported that treatment of human VSMCs³³ and fibroblasts⁴⁰ with PTX failed to inhibit their mitogenic response to PDGF or fibroblast growth factor. Thus, it has been suggested that the effect of PTX is specific to the mitogenic effect of thrombin. We also observed that PTX did not influence PDGF-BB–induced DNA synthesis in rat VSMCs. On the other hand, there is some evidence that thrombin receptors in VSMCs are also able to couple to PTX-insensitive G proteins, which may belong to the phospholipase C–dependent G_q proteins.⁴¹ Findings derived from different cell types also provide evidence that the thrombin receptor may be able to interact with both G_i and G_q proteins.^{42 43} More recently, it has been described that the activated TXA₂ and thrombin receptors in human platelets couple to G proteins of the G₁₂ subfamily and one or more members of the G_q subfamily.⁴⁴

Classic growth factors such as PDGF-BB or epidermal growth factor propagate their mitogenic signals via autophosphorylation of their respective receptors on tyrosine residues.²⁰ Several lines of evidence suggest that in contrast to the Ang II and thrombin receptors, this receptor type is not coupled to G proteins but to ras, a 21-kD protein that belongs to the low molecular weight GTP binding proteins encoded by the ras proto-oncogene involved in PDGF-induced cell proliferation.⁴⁵ In this context, it was suggested that ras functions like other G proteins. The GTPase activity of the ras protein is regulated by the GTPase-activating protein (120 kD). The PDGF-dependent intracellular signal transduction from the receptor into the cell involves different proteins, such as phospholipase C, GTPase-activating protein, and phosphatidylinositol-3 kinase carrying Src homology region 2 (SH2) domains that are capable of binding to specific regions of the receptor containing autophosphorylated tyrosine residues.⁴⁵

Recently, it has been recognized that further transmission of growth signals to the nucleus by growth-promoting factors such as PDGF, thrombin, and Ang II is mediated by sequentially activated protein kinases. The activation of MAP kinases, in particular the 42-kD (p42^mapk) and 44-kD (p44^mapk) isoforms, appears to be a key step in growth signal transduction of several growth-promoting factors, including PDGF,²⁰ Ang II,⁴⁶ and thrombin.⁴⁷ In this context, it is assumed that MAP kinases probably activate S6 protein kinases, in particular the 70-kD (p70^rsk) and 90-kD(p90^rsk) kinases encoded by the rsk gene family. Stimulation of the S6 protein seems to be the requisite for mitogenic signal transduction. Furthermore, it is assumed that activation of MAP kinases is involved in the Ang II–, thrombin-, and PDGF-induced expression of immediate-early genes such as c-fos.^{48 49 50} Recently, the egr-1 gene has also been identified as a transcription factor belonging to the class of immediate-early genes.¹⁷ The c-fos protein is often coregulated with the egr-1 protein. Both are located in the nucleus and play an important role as transcription factors.¹⁷ We have recently demonstrated that Ang II and PDGF are able to induce induction of egr-1 mRNA in VSMCs.^{50 51}

In the present study we were able to show that both CTA₂ and U46619 stimulate the 42-kD MAP kinase isoform and the expression of c-fos and egr-1 mRNA. Similarly to Ang II, thrombin, and PDGF, the TXA₂ mimetics induced a time-dependent stimulation of the 42-kD MAP kinase, with a maximum of 5 to 10 minutes, and a time-dependent increase of c-fos and egr-1 mRNA, with a maximum at 30 minutes. The induction of egr-1 and c-fos by both CTA₂ and U46619 in VSMCs occurred rapidly and transiently, with kinetics similar to those of PDGF,⁵⁰ thrombin,⁴⁹ and Ang II.^{48 51}

Conflicting reports exist about the effects of TXA₂ mimetics on VSMC growth. Dorn et al²⁷ reported that U46619 stimulates c-fos expression and protein synthesis but does not stimulate DNA synthesis. In contrast, Hanasaki et al⁵² reported that U46619 increases [³H]thymidine incorporation and cell proliferation. Similar results were obtained by Ishimitsu et al,⁵ demonstrating that 9,11-epithio-11,12-methano-TXA₂, another stable TXA₂ analogue, can stimulate DNA synthesis in VSMCs from the thoracic aorta of Wistar-Kyoto rats. Dorn et al²⁷ reported that TXA₂/PGH₂ mimetics induce VSMC hypertrophy but not hyperplasia, suggesting a mechanism of action similar to that of Ang II. However, although the predominant opinion of many investigators is that Ang II induces only cellular hypertrophy, defined as an increase in total cellular protein of VSMC but not cellular proliferation (defined as an increase in DNA synthesis with cell division),^{28 29} several studies demonstrate that Ang II induces VSMC proliferation.^{30 31 53 54}

In the present study we demonstrate that both CTA₂ and U46619 induce VSMC proliferation, as determined by [³H]thymidine incorporation into cell DNA and by cell counting. Both agonists stimulate [³H]thymidine incorporation into cell DNA. Furthermore, both substances remarkably enhanced the PDGF-BB–dependent increase in [³H]thymidine incorporation in a synergistic fashion. The TXA₂ mimetic–induced growth signal transduction in VSMCs is mediated by the TXA₂/PGH₂ receptor because SQ29548, a selective TXA₂/PGH₂ receptor antagonist,²¹ effectively blocked the effect of the mimetics on [Ca²⁺]_i and cell DNA synthesis. SQ29548 (10^-6 mol/L) did not affect PDGF-BB–dependent DNA synthesis (data not shown), confirming the high specificity of SQ29548 for the TXA₂ receptor.²¹

Both CTA₂ and U46619 increase cell number and also enhance the PDGF-BB–dependent rise in cell count. In contrast to the findings of Dorn et al,²⁷ the present findings suggest that TXA₂ agonists stimulate DNA synthesis and cell proliferation.

The proliferation of VSMCs in response to various growth factors differs considerably and depends on several factors, such as isolation procedure, strain and age of the animal, cultivating conditions, number of passages, cell seeding density, and culture time.⁵⁵ It has been suggested that the capacity of VSMCs to proliferate depends on the smooth muscle phenotype. Two phenotypes have been discussed: the contractile and synthetic phenotypes. It has been suggested that VSMCs in the contractile state are not able to proliferate.⁵⁵ Changes in smooth muscle phenotype within different passages cannot be excluded. The inconsistencies between our results and those of Dorn et al²⁷ may reflect differences in one or more of these factors.

Obviously, since the concentrations of the TXA₂ mimetics used in our experiments exceed by far the concentrations physiologically occurring in vascular vessels, one has to question the physiological relevance of our findings derived from cultured cells. According to the "response to injury hypothesis" of Ross,⁶ platelets and endothelial cells participate in the development of atheromatous plaque by secretion of PDGF, which is a strong proliferative factor for VSMCs. It can be hypothesized that endothelial injury may be associated with a release of TXA₂ mainly from activated platelets and also from endothelial cells, resulting in an increase of the local concentration of TXA₂. Therefore, TXA₂ alone or in combination with PDGF-BB could enhance VSMC proliferation and thus might contribute to the pathogenesis or progression of cardiovascular diseases. However, the concentrations of the TXA₂ mimetics inducing the observed effects were high compared with TXA₂ mimetic concentrations producing vascular contraction and platelet aggregation. These differences can be explained by the assumption that cultured VSMCs may express less intact TXA₂/PGH₂ receptors than VSMCs in native vessels. Nevertheless, cultured VSMCs are a useful model for studying the effects of growth factors and contractile agonists on their intracellular signaling transduction pathways.

In conclusion, we suggest that the growth-promoting mechanisms of TXA₂/PGH₂ mimetics are very similar to those observed by PDGF, Ang II, and thrombin, including increases in [Ca²⁺]_i, activation of MAP kinase, and expression of the immediate growth-response genes. The growth-promoting effects of TXA₂ mimetics seem to be independent of PTX-sensitive G_i proteins. Our findings are supported by several reports demonstrating that the TXA₂/PGH₂ receptors in platelets and VSMCs are not coupled negatively to the adenylate cyclase via a G_i protein (for review see Reference 56⁵⁶ ). Since the intracellular signal transduction of the TXA₂ mimetics via their TXA₂/PGH₂ receptors in VSMCs is characteristic for receptors that are coupled with G_q proteins, we suggest that the TXA₂/PGH₂ receptors in VSMCs are probably coupled with a G_q protein, as has been observed in platelets with the use of specific antibodies to G_q proteins.^{25 26 56} Finally, we conclude that TXA₂ alone or in combination with PDGF-BB is a powerful stimulator for VSMC growth and therefore might contribute to the pathogenesis of cardiovascular diseases by enhancing VSMC proliferation.

	Selected Abbreviations and Acronyms

 

Ang II = angiotensin II [Ca2+]i = cytosolic free Ca2+ concentration CTA2 = carbocyclic thromboxane A2 DMEM = Dulbecco’s modified Eagle’s medium egr-1 = early growth response gene-1 IP3 = inositol 1,4,5-trisphosphate MAP = mitogen-activated protein PBS = phosphate-buffered saline PDGF = platelet-derived growth factor PGH2 = prostaglandin H2 PTX = pertussis toxin SDS = sodium dodecyl sulfate SDS-PAGE = sodium dodecyl sulfate–polyacrylamide gel electrophoresis TXA2, TXB2 = thromboxane A2, B2 VSMC = vascular smooth muscle cell

	Acknowledgments

 
This work was supported by a grant of the Deutsche Forschungsgemeinschaft (Sa 568/1-1). We are particularly indebted to Vikas P. Sukhatme, Harvard Medical School, Beth Israel Hospital, Boston, Mass, for providing the 2.1-kb egr-1 cDNA probe. We acknowledge helpful discussions with Kerstin Schulte. The excellent technical assistance of Marianne Appenheimer, Claudia Seul, Petra Epping, and Maria-Katharina Meyer zur Brickwedde is greatly appreciated.

Received March 28, 1995; first decision April 13, 1995; accepted June 26, 1995.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Schwartz SM, Reidy M. Common mechanisms of proliferation of smooth muscle in atherosclerosis and hypertension. Hum Pathol. 1987;18:240-247. [Medline] [Order article via Infotrieve]

2. Schwartz SM, Campbell GR, Campbell JH. Replication of smooth muscle cells in vascular disease. Circ Res. 1986;58:427-444. [Abstract/Free Full Text]

3. Morinelli TA, Oatis JE, Okwu AK, Mais DE, Halushka P. Characterization of an ¹²⁵I-labeled thromboxane A₂/prostaglandin H₂ receptor agonist. J Pharmacol Exp Ther. 1989;251:557-562. [Abstract/Free Full Text]

4. Fitzgerald DJ, Roy L, Catella F, Fitzgerald GA. Platelet activation in unstable coronary disease. N Engl J Med. 1986;315:983-989. [Abstract]

5. Ishimitsu T, Uehara Y, Ishi M, Ikeda T, Matsuoka H, Sugimoto T. Thromboxane and vascular smooth muscle cell growth in genetically hypertensive rats. Hypertension. 1988;12:46-51. [Abstract/Free Full Text]

6. Ross R. The pathogenesis of atherosclerosis: an update. N Engl J Med. 1986;314:488-500. [Medline] [Order article via Infotrieve]

7. Dzau VJ, Gibbons GH. Endothelium and growth factors in vascular remodeling of hypertension. Hypertension. 1991;18(suppl III):III-115-III-121.

8. Ross R, Raines EW, Bowen-Pope DF. The biology of platelet-derived growth factor. Cell. 1986;46:155-169. [Medline] [Order article via Infotrieve]

9. Smith EF, Lefer MA, Nicolau KC. Mechanisms of coronary vasoconstriction induced by carbocyclic thromboxane A₂. Am J Physiol. 1981;240:H493-H497.

10. Chamley JH, Campbell GR, Ross R. The smooth muscle cell in culture. Physiol Rev. 1979;39:1-61.

11. Ross RJ. The smooth muscle cell, II: growth of smooth muscle in culture and formation of elastic fiber. J Cell Biol. 1971;50:172-182. [Abstract/Free Full Text]

12. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca²⁺ indicators with greatly improved fluorescence properties. J Biol Chem. 1985;260:3440-3450. [Abstract/Free Full Text]

13. Simm A, Hoppe V, Tatje D, Schenzinger A, Hoppe J. PDGF-AA effectively stimulates early events but has no mitogenic activity in AKR-2B mouse fibroblasts. Exp Cell Res. 1992;201:192-199. [Medline] [Order article via Infotrieve]

14. Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-254. [Medline] [Order article via Infotrieve]

15. Chirgwin JJ, Przybyla RJ, MacDonald RJ, Rutter WJ. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979;18:5294-5299. [Medline] [Order article via Infotrieve]

16. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989;1:7.39-7.52.

17. Sukhatme VP, Cao X, Chang LC, Tsai-Morris CH, Stamenkovich D, Ferreira PCP, Cohen DR, Edwards SA, Shows TB, Curran T, LeBeau MM, Adamson ED. A zinc finger-encoding gene coregulated with c-fos during growth and differentiation and after cellular depolarization. Cell. 1988;53:37-43. [Medline] [Order article via Infotrieve]

18. Sachinidis A, Locher R, Vetter W, Tatje D, Hoppe J. Different effects of PDGF-isoforms on rat vascular smooth muscle cells. J Biol Chem. 1990;265:10238-10243. [Abstract/Free Full Text]

19. Böhm M, Castelano M, Paul M, Erdmann E. Cardiac norepinephrine, ß-adrenoreceptors, and Gi -protein in prehypertensive and hypertensive spontaneously hypertensive rats. J Cardiovasc Pharmacol. 1994;23:980-987. [Medline] [Order article via Infotrieve]

20. Pelech SL, Sanghera S. MAP kinases: charting the regulatory pathways. Science. 1992;257:1355-1356. [Free Full Text]

21. Ogletree ML, Harris DN, Greenberg R, Haslanger MF, Nakane M. Pharmacological action of SQ29548, a novel selective thromboxane antagonist. J Pharmacol Exp Ther. 1985;234:435-441. [Abstract/Free Full Text]

22. Gordeladze JO, Johanson PW, Paulssen RH, Paulssen EJ, Gautvik KM. G-proteins: implication for pathophysiology and disease. Eur J Endocrinol. 1994;131:557-574. [Abstract/Free Full Text]

23. Hepler JR, Gilman AG. G proteins. Trends Biochem Sci. 1992;17:383-387. [Medline] [Order article via Infotrieve]

24. Sternweis PC, Smrcka AV. Regulation of phospholipase C by G proteins. Trends Biochem Sci. 1992;17:502-505. [Medline] [Order article via Infotrieve]

25. Shenker A, Goldsmith P, Unson CG, Spiegel AM. The G protein coupled to the TXA₂/PGH₂ receptor in human platelets is a member of the novel Gq family. J Biol Chem. 1991;266:9309-9313. [Abstract/Free Full Text]

26. Knezewic T, Borg C, Le Brelon GC. Identification of Gq as one of the G-proteins which copurify with human platelet thromboxane A₂/prostaglandin H₂ receptors. J Biol Chem. 1993;268:26111-26117.

27. Dorn GW, Becker MW, Davis M. Dissociation of the contractile and hypertrophic effects of vasoconstrictor prostanoids in vascular smooth muscle cells. J Biol Chem. 1992;267:24897-24905. [Abstract/Free Full Text]

28. Berk BC, Vekshtein V, Gordon HM, Tsuda T. Angiotensin II-stimulated protein synthesis in cultured vascular smooth muscle cells. Hypertension. 1989;13:305-314. [Abstract/Free Full Text]

29. Turla MB, Thompson MM, Corjay MH, Owens GK. Mechanisms of angiotensin II– and arginine vasopressin–induced increases in protein synthesis and content in cultured rat aortic smooth muscle cells. Hypertension. 1991;68:288-299.

30. Sachinidis A, Ko Y, Nettekoven W, Wieczorek AJ, Düsing R, Vetter H. The effect of angiotensin II on DNA synthesis varies considerably in vascular smooth muscle cells from different Wistar-Kyoto rats. J Hypertens. 1992;10:1159-1164. [Medline] [Order article via Infotrieve]

31. Daemen MJAP, Lombardi DM, Bosman FT, Schwartz SM. Angiotensin II induces smooth muscle cell proliferation in the normal and injured rat arterial wall. Circ Res. 1991;68:450-456. [Abstract/Free Full Text]

32. Stouffer GA, Sarembrock IJ, McNamara CA, Gimple LW, Owens G. Thrombin-induced mitogenesis of vascular SMC is partially mediated by autocrine production of PDGF-AA. Am J Physiol. 1993;265:C806-C811. [Abstract/Free Full Text]

33. Kanthou C, Parry G, Wijelath E, Kakkar VV, Demoliou-Mason C. Thrombin-induced proliferation and expression of platelet-derived growth factor-A chain gene in human vascular smooth muscle cells. FEBS Lett. 1992;314:143-148. [Medline] [Order article via Infotrieve]

34. Griendling KK, Rittenhouse SE, Brock TA, Ekstein LS, Gimbrone MA Jr, Alexander RW. Sustained diacylglycerol formation from inositol phospholipids in angiotensin II-stimulated vascular smooth muscle cells. J Biol Chem. 1986;261:5901-5906. [Abstract/Free Full Text]

35. Huang C-H, Cogan MG, Cragoe EJ, Ives H. Thrombin activation of the Na⁺/H⁺ exchanger in vascular smooth muscle cells. J Biol Chem. 1987;262:14134-14140. [Abstract/Free Full Text]

36. Rozengurt E. Early signals in the mitogenic response. Science. 1986;234:161-166. [Abstract/Free Full Text]

37. Gutowski S, Smrcka A, Nowak L, Wu D, Simon M, Sternweis PC. Antibodies to the q subfamily of guanine nucleotide-binding regulatory protein subunits attenuate activation of phosphatidylinositol 4,5-bisphosphate hydrolysis by hormones. J Biol Chem. 1991;266:20519-20524. [Abstract/Free Full Text]

38. Bihoreau C, Monnot C, Davies E, Teutsch B, Bernstein K, Corvol P, Clauser E. Mutation of Asp74 of the rat angiotensin II receptor confers changes in antagonist affinities and abolishes G-protein coupling. Proc Natl Acad Sci U S A. 1993;90:5133-5137. [Abstract/Free Full Text]

39. Socorro L, Alexander RW, Griendling KK. Cholera toxin modulation of angiotensin II-stimulated inositol phosphate production in cultured vascular smooth muscle cells. Biochem J. 1990;265:799-807. [Medline] [Order article via Infotrieve]

40. Chambard CJ, Paris S, L’Allemain G, Pouysségur J. Two growth signalling pathways in fibroblasts distinguished by pertussis toxin. Nature. 1987;326:800-803. [Medline] [Order article via Infotrieve]

41. Neylon C, Nickashin A, Little PJ, Tkachuk V, Bobik A. Thrombin-induced Ca²⁺ mobilization in vascular smooth muscle utilizes a slowly ribosylating pertussis toxin-sensitive G protein. J Biol Chem. 1992;267:7295-7302. [Abstract/Free Full Text]

42. Vouret-Craviary V, Van Obberghen-Schilling E, Rasmusen UB, Paviariny A, Lecocq JP, Pouysségur J. Synthetic alpha-thrombin receptor peptides activate G protein-coupled signaling pathways but are unable to induce mitogenesis. Mol Biol Cell. 1992;3:95-102. [Abstract]

43. Simon MI, Stratmann MP, Gautam N. Diversity of signal transduction. Science. 1991;252:802-808. [Abstract/Free Full Text]

44. Offermans S, Laugwitz KL, Spicher K, Schulz G. G proteins of the G12 family are activated via the thromboxane A₂ and thrombin receptors in human platelets. Proc Natl Acad Sci U S A. 1994;91:504-508. [Abstract/Free Full Text]

45. Kaplan DR, Morrison DK, Wong G, McCormick F, Williams L. PDGF ß-receptor stimulates tyrosine phosphorylation of GAP and association of GAP with a signaling complex. Cell. 1990;61:125-133. [Medline] [Order article via Infotrieve]

46. Duff JL, Berk BC, Corson MA. Angiotensin II stimulates the pp44 and pp42 mitogen-activated protein kinases in cultured rat aortic smooth muscle cells. Biochem Biophys Res Commun. 1992;188:257-264. [Medline] [Order article via Infotrieve]

47. L’Allemain G, Pouyssegur J, Weber MJ. p42/mitogen-activated protein kinase as a converging target for different growth factor signaling pathway: use of pertussis toxin as a discrimination factor. Cell Regul. 1991;2:675-684. [Medline] [Order article via Infotrieve]

48. Taubman MB, Berk BC, Izumo S, Tsuda T, Alexander RW, Nadal-Ginard B. Angiotensin II induces c-fos mRNA in aortic smooth muscle. J Biol Chem. 1989;264:526-530. [Abstract/Free Full Text]

49. Berk BC, Taubman MB, Cragoe EJ Jr, Fenton JW II, Griendling KK. Thrombin signal transduction mechanisms in rat vascular smooth muscle cells. J Biol Chem. 1990;265:17334-17340. [Abstract/Free Full Text]

50. Sachinidis A, Schulte K, Ko Y, Meyer zu Brickwedde K, Hoppe V, Hoppe J, Vetter H. The induction of early response gene in rat smooth muscle cells by PDGF-AA is not sufficient to stimulate DNA-synthesis. FEBS Lett. 1993;319:221-224. [Medline] [Order article via Infotrieve]

51. Sachinidis A, Weisser P, Ko Y, Schulte K, Meyer zu Brickwedde K, Neyses L, Vetter H. Angiotensin II induces formation of the early growth response gene-1 protein in rat vascular smooth muscle cells. FEBS Lett. 1992;313:109-112. [Medline] [Order article via Infotrieve]

52. Hanasaki K, Nakano T, Arita H. Receptor-mediated mitogenic effect of thromboxane A2 in vascular smooth muscle cells. Biochem Pharmacol. 1990;40:2535-2542. [Medline] [Order article via Infotrieve]

53. Weissberg PL, Grainger DJ, Shanahan C, Metcalfe J. Approaches to the development of selective inhibitors of vascular smooth muscle cell proliferation. Cardiovasc Res. 1993;27:1191-1198. [Free Full Text]

54. Bagby SP, Kirk EA, Mitchell LH, O’Reilly MM, Holden WE, Stenberg PE, Bakke A. Proliferative synergy of Ang II and EGF in porcine aortic vascular smooth muscle cells. Am J Physiol. 1993;265:F239-F249. [Abstract/Free Full Text]

55. Campbell JH, Campbell RG. Culture techniques and their applications to studies of vascular smooth muscle. Clin Sci. 1993;85:501-513. [Medline] [Order article via Infotrieve]

56. Coleman RA, Smith WL, Narumya S. VIII International Union of Pharmacology Classification of prostanoid receptors: properties, distribution, and structure of the receptors and their subtypes. Pharmacol Rev. 1994;46:205-229.[Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				C.-S. Lee, Y.-W. Kwon, H.-M. Yang, S.-H. Kim, T.-Y. Kim, J. Hur, K.-W. Park, H.-J. Cho, H.-J. Kang, Y.-B. Park, et al. New Mechanism of Rosiglitazone to Reduce Neointimal Hyperplasia: Activation of Glycogen Synthase Kinase-3{beta} Followed by Inhibition of MMP-9 Arterioscler. Thromb. Vasc. Biol., April 1, 2009; 29(4): 472 - 479. [Abstract] [Full Text] [PDF]

				H. Obata, Y. Sakai, S. Ohnishi, S. Takeshita, H. Mori, M. Kodama, K. Kangawa, Y. Aizawa, and N. Nagaya Single Injection of a Sustained-release Prostacyclin Analog Improves Pulmonary Hypertension in Rats Am. J. Respir. Crit. Care Med., January 15, 2008; 177(2): 195 - 201. [Abstract] [Full Text] [PDF]

				F. Michel, J.-S. Silvestre, L. Waeckel, S. Corda, T. Verbeuren, J. P. Vilaine, M. Clergue, M. Duriez, and B. I. Levy Thromboxane A2/Prostaglandin H2 Receptor Activation Mediates Angiotensin II-Induced Postischemic Neovascularization Arterioscler. Thromb. Vasc. Biol., March 1, 2006; 26(3): 488 - 493. [Abstract] [Full Text] [PDF]

				H. S. Jung, K.-H. Park, Y. M. Cho, S. S. Chung, H. J. Cho, S. Y. Cho, S. J. Kim, S. Y. Kim, H. K. Lee, and K. S. Park Resistin is secreted from macrophages in atheromas and promotes atherosclerosis Cardiovasc Res, January 1, 2006; 69(1): 76 - 85. [Abstract] [Full Text] [PDF]

				D. J. Rademacher, S. Patel, W.-S. V. Ho, A. M. Savoie, N. J. Rusch, K. M. Gauthier, and C. J. Hillard U-46619 but not serotonin increases endocannabinoid content in middle cerebral artery: evidence for functional relevance Am J Physiol Heart Circ Physiol, June 1, 2005; 288(6): H2694 - H2701. [Abstract] [Full Text] [PDF]

				H.-M. Yang, H.-S. Kim, K.-W. Park, H.-J. You, S.-I. Jeon, S.-W. Youn, S.-H. Kim, B.-H. Oh, M.-M. Lee, Y.-B. Park, et al. Celecoxib, a Cyclooxygenase-2 Inhibitor, Reduces Neointimal Hyperplasia Through Inhibition of Akt Signaling Circulation, July 20, 2004; 110(3): 301 - 308. [Abstract] [Full Text] [PDF]

				H. Francois, K. Athirakul, L. Mao, H. Rockman, and T. M. Coffman Role for Thromboxane Receptors in Angiotensin-II-Induced Hypertension Hypertension, February 1, 2004; 43(2): 364 - 369. [Abstract] [Full Text] [PDF]

				A. Haider, I. Lee, J. Grabarek, Z. Darzynkiewicz, and N. R. Ferreri Dual Functionality of Cyclooxygenase-2 as a Regulator of Tumor Necrosis Factor-Mediated G1 Shortening and Nitric Oxide-Mediated Inhibition of Vascular Smooth Muscle Cell Proliferation Circulation, August 26, 2003; 108(8): 1015 - 1021. [Abstract] [Full Text] [PDF]

				T. J. Weber and L. M. Markillie Regulation of Activator Protein-1 by 8-iso-Prostaglandin E2 in a Thromboxane A2 Receptor-Dependent and -Independent Manner Mol. Pharmacol., May 1, 2003; 63(5): 1075 - 1081. [Abstract] [Full Text] [PDF]

				S. Brault, A. K. Martinez-Bermudez, A. M. Marrache, F. Gobeil Jr, X. Hou, M. Beauchamp, C. Quiniou, G. Almazan, C. Lachance, J. Roberts II, et al. Selective Neuromicrovascular Endothelial Cell Death by 8-Iso-Prostaglandin F2{alpha}: Possible Role in Ischemic Brain Injury Stroke, March 1, 2003; 34(3): 776 - 782. [Abstract] [Full Text] [PDF]

				I. GOUNI-BERTHOLD and A. SACHINIDIS Does the coronary risk factor low density lipoprotein alter growth and signaling in vascular smooth muscle cells? FASEB J, October 1, 2002; 16(12): 1477 - 1487. [Abstract] [Full Text] [PDF]

				M. Katsuyama, C. Fan, and C. Yabe-Nishimura NADPH Oxidase Is Involved in Prostaglandin F2alpha -induced Hypertrophy of Vascular Smooth Muscle Cells. INDUCTION OF NOX1 BY PGF2alpha J. Biol. Chem., April 12, 2002; 277(16): 13438 - 13442. [Abstract] [Full Text] [PDF]

				C. D. Fike, S. L. Pfister, M. R. Kaplowitz, and J. A. Madden Cyclooxygenase contracting factors and altered pulmonary vascular responses in chronically hypoxic newborn pigs J Appl Physiol, January 1, 2002; 92(1): 67 - 74. [Abstract] [Full Text] [PDF]

				H. V. Anderson, J. McNatt, F. J. Clubb, M. Herman, J.-P. Maffrand, F. DeClerck, C. Ahn, L. M. Buja, and J. T. Willerson Platelet Inhibition Reduces Cyclic Flow Variations and Neointimal Proliferation in Normal and Hypercholesterolemic-Atherosclerotic Canine Coronary Arteries Circulation, November 6, 2001; 104(19): 2331 - 2337. [Abstract] [Full Text] [PDF]

				I. Gouni-Berthold, C. Seul, Y. Ko, J. Hescheler, and A. Sachinidis Gangliosides GM1 and GM2 Induce Vascular Smooth Muscle Cell Proliferation via Extracellular Signal-Regulated Kinase 1/2 Pathway Hypertension, November 1, 2001; 38(5): 1030 - 1037. [Abstract] [Full Text] [PDF]

				J. Jankowski, A. Schroter, M. Tepel, M. van der Giet, N. Stephan, J. Luo, W. Zidek, and H. Schluter Isolation and Characterization of Coenzyme A Glutathione Disulfide as a Parathyroid-Derived Vasoconstrictive Factor Circulation, November 14, 2000; 102(20): 2548 - 2552. [Abstract] [Full Text] [PDF]

				H. Kraiczi, J. Hedner, Y. Peker, and J. Carlson Increased vasoconstrictor sensitivity in obstructive sleep apnea J Appl Physiol, August 1, 2000; 89(2): 493 - 498. [Abstract] [Full Text] [PDF]

				W. Young, K. Mahboubi, A. Haider, I. Li, and N. R. Ferreri Cyclooxygenase-2 Is Required for Tumor Necrosis Factor-{alpha}- and Angiotensin II-Mediated Proliferation of Vascular Smooth Muscle Cells Circ. Res., April 28, 2000; 86(8): 906 - 914. [Abstract] [Full Text] [PDF]

				T. J. Weber, T. J. Monks, and S. S. Lau DDM-PGE2-mediated cytoprotection in renal epithelial cells by a thromboxane A2 receptor coupled to NF-kappa B Am J Physiol Renal Physiol, February 1, 2000; 278(2): F270 - F278. [Abstract] [Full Text] [PDF]

				R. Pakala, R. Pakala, W. L. Sheng, and C. R. Benedict Eicosapentaenoic Acid and Docosahexaenoic Acid Block Serotonin-Induced Smooth Muscle Cell Proliferation Arterioscler. Thromb. Vasc. Biol., October 1, 1999; 19(10): 2316 - 2322. [Abstract] [Full Text] [PDF]

				A. Sachinidis, R. Kettenhofen, S. Seewald, I. Gouni-Berthold, U. Schmitz, C. Seul, Y. Ko, and H. Vetter Evidence That Lipoproteins Are Carriers of Bioactive Factors Arterioscler. Thromb. Vasc. Biol., October 1, 1999; 19(10): 2412 - 2421. [Abstract] [Full Text] [PDF]

				H.-Y. Ahn, K. R. Hadizadeh, C. Seul, Y.-P. Yun, H. Vetter, and A. Sachinidis Epigallocathechin-3 Gallate Selectively Inhibits the PDGF-BB-induced Intracellular Signaling Transduction Pathway in Vascular Smooth Muscle Cells and Inhibits Transformation of sis-transfected NIH 3T3 Fibroblasts and Human Glioblastoma Cells (A172) Mol. Biol. Cell, April 1, 1999; 10(4): 1093 - 1104. [Abstract] [Full Text]

				M. Camacho, J. Lopez-Belmonte, and L. Vila Rate of Vasoconstrictor Prostanoids Released by Endothelial Cells Depends on Cyclooxygenase-2 Expression and Prostaglandin I Synthase Activity Circ. Res., August 24, 1998; 83(4): 353 - 365. [Abstract] [Full Text] [PDF]

				S. Seewald, C. Seul, R. Kettenhofen, D. Bokemeyer, Y. Ko, H. Vetter, and A. Sachinidis Role of Mitogen-Activated Protein Kinase in the Angiotensin II–Induced DNA Synthesis in Vascular Smooth Muscle Cells Hypertension, May 1, 1998; 31(5): 1151 - 1156. [Abstract] [Full Text] [PDF]

				C. K. Fujihara, D. M. A. C. Malheiros, J. L. Donato, A. Poli, G. De Nucci, and R. Zatz Nitroflurbiprofen, a new nonsteroidal anti-inflammatory, ameliorates structural injury in the remnant kidney Am J Physiol Renal Physiol, March 1, 1998; 274(3): F573 - F579. [Abstract] [Full Text] [PDF]

				T.-Ph. Zucker, D. Bonisch, S. Muck, A.-A. Weber, E. Bretschneider, E. Glusa, and K. Schror Thrombin-Induced Mitogenesis in Coronary Artery Smooth Muscle Cells Is Potentiated by Thromboxane A2 and Involves Upregulation of Thromboxane Receptor mRNA Circulation, February 17, 1998; 97(6): 589 - 595. [Abstract] [Full Text] [PDF]

				R. Pakala, J. T. Willerson, and C. R. Benedict Effect of Serotonin, Thromboxane A2, and Specific Receptor Antagonists on Vascular Smooth Muscle Cell Proliferation Circulation, October 7, 1997; 96(7): 2280 - 2286. [Abstract] [Full Text]

				S. Seewald, G. Nickenig, Y. Ko, H. Vetter, and A. Sachinidis Low density lipoprotein enhances the thrombin-induced growth of vascular smooth muscle cells Cardiovasc Res, October 1, 1997; 36(1): 92 - 100. [Abstract] [Full Text] [PDF]

				A. Sachinidis, S. Seewald, P. Epping, C. Seul, Y. Ko, and H. Vetter The Growth-Promoting Effect of Low-Density Lipoprotein May Be Mediated by a Pertussis Toxin-Sensitive Mitogen-Activated Protein Kinase Pathway Mol. Pharmacol., September 1, 1997; 52(3): 389 - 397. [Abstract] [Full Text]

				S. L. Pfister, D. A. Kotulock, and W. B. Campbell Vascular Smooth Muscle Thromboxane A2 Receptors Mediate Arachidonic Acid-Induced Sudden Death in Rabbits Hypertension, January 1, 1997; 29(1): 303 - 309. [Abstract] [Full Text] [PDF]

				A. Sachinidis, M. Liu, A.-A. Weber, C. Seul, V. Harth, S. Seewald, Y. Ko, and H. Vetter Cholesterol Enhances Platelet-Derived Growth Factor-BB-Induced [Ca2+]i and DNA Synthesis in Rat Aortic Smooth Muscle Cells Hypertension, January 1, 1997; 29(1): 326 - 333. [Abstract] [Full Text] [PDF]

This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sachinidis, A. Articles by Vetter, H. Search for Related Content PubMed PubMed Citation Articles by Sachinidis, A. Articles by Vetter, H. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB CALCIUM COMPOUNDS CALCIUM, ELEMENTAL