Different Effects of Thrombin Receptor Activation on Endothelium and Smooth Muscle Cells of Human Coronary Bypass Vessels
Implications for Venous Bypass Graft Failure
Background Thrombin is implicated in coronary bypass graft disease; it cleaves its receptor’s extracellular N-terminal domain and unmasks a new N-terminus as a tethered ligand. We studied the effects of thrombin receptor activation in human internal mammary artery (IMA) and saphenous vein (SV).
Methods and Results To study the effects of thrombin receptor activation on vasomotion, isolated blood vessels were suspended for isometric tension recording, and the effects on cell proliferation were studied in cultured smooth muscle cells (SMCs) of IMA and SV. Thrombin receptor expression in IMA and SV was analyzed by reverse transcription polymerase chain reaction and immunohistology. Receptor function was studied by analyzing the activation of mitogen-activated protein kinase (p42MAPK). In IMA thrombin evoked endothelium-dependent relaxations (65±5%) that were mimicked by thrombin receptor agonist peptide (TRAP) and reduced by the thrombin inhibitors recombinant (r-) hirudin and d-Phe-Pro-Arg-chloromethyl ketone (PPACK) (P<.05). In SV thrombin caused contractions (36±5% of 100 mmol/L KCl) that were inhibited by r-hirudin or PPACK (P<.05) but not mimicked by TRAP. In SMCs thrombin induced more pronounced [3H]thymidine incorporation (inhibited by r-hirudin or PPACK) in SV than IMA (P<.05), but activation of p42MAPK was similar in both vessels. TRAP induced weaker activation of p42MAPK than thrombin and did not stimulate [3H]thymidine incorporation in SMCs of SV or IMA. Immunohistology and RT-PCR demonstrated that the endothelium and SMCs of IMA and SV express thrombin receptor.
Conclusions Functional thrombin receptors are present on endothelium and SMCs of IMA and SV. Endothelial thrombin receptors mediate relaxation in IMA but not SV. Thrombin causes much more pronounced contraction and proliferation in SMCs of SV than IMA independent of tethered receptors, suggesting other thrombin receptors exist. These differences of thrombin receptor activation in IMA and SV may be important in the development of and therapy for graft disease.
Previous studies have demonstrated the lower patency rate of SV bypass grafts compared with IMA grafts.1 This may be due to different endothelial production of vasodilators (ie, NO and prostacyclin) and platelet–vessel wall interactions2 3 as well as different contractile and proliferative properties of SMCs.4 5 Thrombin is an important modulator of all these events,6 and antithrombotic therapy improves patency and clinical events in patients receiving venous grafts.7
Thrombin affects a wide range of cell types, such as platelets, monocytes, ECs, and SMCs,2 8 9 10 11 and hence is an important regulator of platelet–vessel wall interaction.12 Indeed, thrombin stimulates platelets to release large amounts of vasoconstrictors (ie, thromboxane A2 and serotonin12 13 ), which in turn causes profound vasoconstriction and platelet aggregation. On the other hand, thrombin activates the ECs of certain blood vessels to release NO and prostacyclin, both of which antagonize vascular contraction and inhibit platelet aggregation.12 14 Thrombin also stimulates SMC contraction and proliferation in animals11 15 16 and is believed to play an important role in vasospasm and neointimal formation.17 18
A human thrombin receptor has been cloned19 that is a member of the seven-transmembrane domain receptor family coupled to guanine nucleotide–binding protein (G protein).19 Thrombin activates its receptor by an unprecedented mechanism in which the serine protease cleaves the receptor’s N-terminal extracellular domain, generating a new N-terminus that then functions as a tethered peptide ligand and activates the receptor.19 A homologous synthetic peptide (SFLLRNPNDKYEPF) corresponding to the tethered ligand sequence of the human thrombin receptor (ie, TRAP) can mimic specific receptor activation under some, but not all, conditions.11 19 20 21 22 Signal transduction studies have demonstrated that thrombin receptor stimulation induces an array of intracellular events,23 24 such as activation of MAPKs 24 25 26 and a pathway activated by growth factor tyrosine kinase receptors such as platelet-derived growth factor receptor.27 This signaling mechanism has been proposed as one of the major pathways for transmitting extracellular stimuli to the cell nucleus regulating cell proliferation27 and is essential for the growth of CCL39 cells.28
The thrombin receptor is expressed in ECs and vascular SMCs as well as platelets.15 19 In experimental animals it is upregulated in SMCs immediately after vascular injury and throughout neointimal development,29 a process that occurs during and after venous bypass implantation.2 30 31 We investigated different effects of thrombin receptor activation on endothelium (ie, endothelium-dependent modulation of vascular tone) and SMC function (contraction and proliferation) of human IMA and SV. Such differences might be of great biological and clinical interest as they may contribute to the different functions and patencies of arterial and venous bypass grafts.
Chemicals and Materials
Acetylcholine hydrochloride, bradykinin, l-norepinephrine bitartrate, thrombin, TRAP, and r-hirudin (activity, 6000 U/mg; 1 U neutralizes 1 U=3×10−8 mol/L of thrombin) were from Sigma. PPACK was from Calbiochem. All materials for cell culture were from GIBCO BRL. The concentrations of the drugs are expressed as final concentrations in the organ-chamber solution or in cell-culture medium. All drugs were dissolved in distilled water for organ-chamber or in PBS for cell-culture experiments except PPACK, which was dissolved in 50 mmol/L sodium acetate and 1% BSA (pH 4.5) at a stock solution of 10−2 mol/L and diluted further in PBS.
Assessment of Vasomotion
IMAs and SVs were obtained intraoperatively.2 3 4 After the vessels were removed, they were placed in modified Krebs-Ringer bicarbonate solution at 4°C (in mmol/L): NaCl 118, KCl 4.7, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25.0, edetate calcium disodium 0.026, and glucose 11.1. The blood vessels were dissected free, cut into 5-mm-long rings, and suspended in organ chambers filled with the solution described above at 37°C (95% O2/5% CO2); changes in isometric tension were recorded with force transducers (Statham Universal UC2).
Prior to the experiment, the rings were stretched and repeatedly exposed to norepinephrine (3×10−7 mol/L for arteries and 10−7 mol/L for veins) until the optimal length-tension relationship was reached4 32 ; they were then allowed to equilibrate for 45 minutes. The endothelium was removed from some rings by gentle rubbing of the intraluminal surface with a cotton swab. The presence or absence of the endothelium was confirmed by the presence or absence of a relaxation to acetylcholine (10−6 mol/L; IMA) or bradykinin (10−6 mol/L; SV).4 32
To study endothelium-dependent responses to thrombin and TRAP, experiments were performed in parallel rings with or without endothelium of arteries or veins contracted with norepinephrine (3×10−7 or 10−7 mol/L, respectively). Thrombin (3×10−8 mol/L), TRAP (10−5 mol/L), thrombin treated with r-hirudin (5 U/mL; activity, 6000 U/mg; 1 U neutralizes 1 U=3×10−8 mol/L of thrombin), or PPACK (10−6 mol/L) for 15 minutes at 37°C were added on top of that contraction. This concentration of thrombin exerts maximal effects.12 Because of the frequent development of tachyphylaxis to thrombin, concentration-response curves were avoided. To study the direct contractile effects of thrombin and TRAP on SMCs, the agents were added to the quiescent rings with or without endothelium of both arteries and veins.
Cultivation of ECs and SMCs
ECs were isolated from HUVECs, SVs, and IMAs.33 Briefly, fresh blood vessels were harvested in cold, sterile RPMI 1640 medium with 100 U/mL penicillin and 100 μg/mL streptomycin. The vessels, cleaned of connective tissue and adventitia, were incubated with collagenase type II (75 U/mL) for 15 minutes in PBS. Cell pellets were then collected by centrifugation at 1000 rpm for 10 minutes, seeded in culture dishes coated with 25 μg/mL human fibronectin, and cultured in RPMI 1640 supplemented with 20 mmol/L l-glutamine, HEPES buffer solution, 100 U/mL penicillin, 100 μg/mL streptomycin, 50 μg/mL EC growth supplement, 25 μg/mL heparin, and 20% FCS. The next day cells were washed with the medium to eliminate blood cell contamination. ECs were identified by their typical cobblestone and nonoverlapping appearance as well as by indirect immunofluorescence staining using specific antibodies against von Willebrand factor. Cells from the third passage were used.
Vascular SMCs were cultivated from SVs and IMAs obtained from patients undergoing coronary bypass surgery using an explant technique.5 34 Briefly, the cells were cultured in DMEM containing 20% FCS supplemented with 20 mmol/L l-glutamine and 10 mmol/L HEPES buffer solution, 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified atmosphere (95% air/5% CO2) at 37°C. Culture medium was replaced every 3 days. Cells were passaged by cell dissociation solution. Experiments were performed on passages 3 through 6. SMCs were characterized by their typical morphological pattern (multilayer sheets and “hills and valleys”) and by indirect immunofluorescence staining using specific mouse monoclonal antibodies against human SMC α-actin (Sigma).5 34
SMCs in DMEM containing 20% FCS were seeded on 12-well plates (density, 2×104/mL) for 24 hours to allow attachment. Culture media were then replaced with serum-free DMEM containing all ingredients described above except for 0.2% BSA instead of FCS. Cells in serum-free DMEM medium were incubated for 48 hours to achieve quiescence; the cells were then stimulated with thrombin (1.5×10−8 to 1.5×10−7 mol/L), TRAP (10−7 to 10−5 mol/L), thrombin treated with r-hirudin (0.5 to 5 U/mL), or PPACK (10−6 to 10−5 mol/L) as described above. [3H]thymidine incorporation was assayed after 24 hours of stimulation.5 34 Incorporated radioactivity was measured by using a β-counter (ICN, Intertechnique).
p42MAPK activation (phosphorylation) by thrombin or TRAP was analyzed by mobility shifts on Western blots. Quiescent SMCs that had rested in serum-free medium for 48 hours were stimulated with thrombin (3×10−8 mol/L) or TRAP (10−5 mol/L) at different times as indicated in “Results.” After being stimulated, the cells were washed immediately with ice-cold PBS and harvested with cold extraction buffer (120 mmol/L sodium chloride, 50 mmol/L Tris, 20 mmol/L sodium fluoride, 1 mmol/L benzamidine, 1 mmol/L dithiothreitol, 1 mmol/L EDTA, 6 mmol/L EGTA, 15 mmol/L sodium pyrophosphate, 0.8 μg/mL leupeptin, 30 mmol/L p-nitrophenyl phosphate, 0.1 mmol/L phenylmethylsulfonyl fluoride, and 1% Nonidet P40). The cell debris was removed by centrifugation at 12 000g for 10 minutes at 4°C. The samples (20 μg) were treated with 5× Laemmli’s SDS-PAGE sample buffer (0.35 mol/L Tris-Cl, pH 6.8, 15% SDS, 56.5% glycerol, and 0.0075% bromophenol blue), heated at 95°C for 3 minutes, and subjected to a 10% SDS-PAGE gel for electrophoresis. The proteins were then transferred to Immobilon-P filter papers (Millipore AG) by using a semidry transfer unit (Hoefer Scientific Instruments). The membranes were blocked by using 5% skim milk in PBS-Tween buffer (0.1% Tween 20; pH=7.5) for 1 hour and incubated with anti-p42MAPK antibodies (1:3000 for MK12 [GIBCO BRL] or 1:1000 for C14 [Santa Cruz Biotech]). The immunoreactive bands were detected by using an enhanced chemiluminescence system (Amersham).
Thrombin Receptor Analyses
Total RNA was isolated from cultured SMCs and ECs of IMA and SV and HUVECs (as positive control) by using TRIzol reagent (GIBCO BRL) according to the manufacturer’s instructions. cDNA was produced from these RNA samples by RT [Superscrip (R)-reverse transcriptase] of 4 μg total RNA from each sample. The cDNA encoding the thrombin receptor was amplified by using PCR with the two primers 5′-TGTGAACTGATCATGTTTATG-3′ and 5′-TTCGTAAGATAAGAGATATGT-3′19 (MWG Biotech). PCR was then performed for 30 cycles (denaturing at 97°C for 15 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 60 seconds). Specific amplification of thrombin receptor cDNA with these primers should yield a 708-bp product.19 Control PCR was performed without RT or with templates from the same RNA isolation with the primers 5′-CAGGAATTCGGTGAAGGTCGGAGTCAACGG-3′ and 5′-AGTGGATCCGGTCATGAGTCCTTCCACGAT-3′ (MWG Biotech) for the housekeeping gene GAPDH (20 cycles). Specific amplification of GAPDH with these primers yielded a 517-bp product as expected. PCR products were then separated by 1% agarose gel electrophoresis and visualized using the Visionary (R) gel documentation system (Bio Cell Consulting).
IMAs and SVs were harvested, dissected free, frozen immediately in liquid nitrogen after removal, and kept at −70°C until used. Serial 6-μm frozen sections of IMAs and SVs were cut in a cryostat (−20°C) and mounted on clean glass slides. After air drying, the slides were fixed in paraformaldehyde according to routine procedures. To block nonspecific binding the slides were soaked in TBS and then incubated in 4% human albumin (Behringwerke AG) solution in TBS for 30 minutes at room temperature. Sections were then incubated for 60 minutes at room temperature with or without a polyclonal rabbit thrombin receptor antibody (kindly provided by Dr H. Theunissen, Department of Biotechnology and Biochemistry, Organon, Netherlands) raised against TRAP, the new N-terminus peptide sequence of thrombin receptor (1:10). The sections were then washed three times for 15 minutes with TBS-Tween (0.05% Tween 20) followed by incubation with biotinylated goat anti-rabbit secondary antibody (Vector Laboratories) for 1 hour at room temperature. After washing with TBS-Tween as described above, the slides were incubated with avidin-biotin–horseradish peroxidase complex. After washing with TBS-Tween as above, bound peroxidase was detected by incubation with the chromogenic substrate 3,3′ diaminobenzidine (Sigma; 50 mg in 100 mL of 0.1 mol/L PBS, to which was added 33 μL H2O2, pH=7.35). After 10 minutes, the slides were rinsed in deionized water, dehydrated, and mounted.
Data are given as mean±SEM. Contractions are expressed as percent of the increase in tension to 100 mmol/L KCl. Relaxations are expressed as percent decrease in tension of the contraction to norepinephrine. Stimulated [3H]thymidine incorporation is expressed as percent increase above control. In all experiments, n equals the number of patients from whom blood vessels were obtained. Student’s t test for paired observations and ANOVA followed by Scheffé’s test for repeated measurements were used for statistical analysis. A two-tailed probability value <.05 was considered significant.
Thrombin and Endothelium-Dependent Relaxations
In IMAs with endothelium contracted with norepinephrine (3×10−7 mol/L), thrombin (3×10−8 mol/L) induced a potent endothelium-dependent relaxation (65±5%, n=6; Fig 1⇓). In contrast, in the SVs, even in the presence of the endothelium, thrombin evoked a small contraction (13±20%, n=6, P<.05 versus artery; Fig 1⇓).
In IMAs with endothelium contracted with norepinephrine (3×10−7 mol/L), TRAP (10−5 mol/L) induced endothelium-dependent relaxations (53±6%, n=4; Fig 2⇓) that were comparable to thrombin-induced relaxations (3×10−8 mol/L, NS). The relaxations to thrombin (43±6%, n=12) were markedly reduced by the thrombin inhibitors r-hirudin (5 U/mL; 12±3%, n=5) and PPACK (10−6 mol/L; 7±2%, n=5, P<.05 versus thrombin).
Thrombin and Vascular Contractions
In quiescent IMAs with or without endothelium, thrombin (3×10−8 mol/L) had no effects on vascular tone (n=8). In contrast, in quiescent SVs with ECs, thrombin (3×10−8 mol/L) induced a pronounced contraction (36±5% of 100 mmol/L KCl, n=11) that was markedly inhibited by 5 U/mL r-hirudin (4.5±1%, n=9, P<.05 versus control) and 10−6 mol/L PPACK (1±0.6%, n=8, P<.05 versus control) (Fig 3⇓). The contractile effects of thrombin in the vein were not affected by removal of the endothelium (data not shown; n=18).
The contractions induced by thrombin in quiescent SVs could only be partially mimicked by TRAP. Indeed, TRAP at a high concentration (10−5 mol/L) induced only a small contraction compared with thrombin (5.9±1%, n=6, P<.05 versus thrombin) (Fig 3⇑).
Thrombin and MAPK Activation
In cultured SVs as well as IMA SMCs, thrombin (3×10−8 mol/L) stimulated p42MAPK phosphorylation in a time-dependent manner as demonstrated by the slower mobility of the phosphorylated form on Western blots. The activation of p42MAPK started at 2 minutes and reached the maximum at 5 to 10 minutes; dephosphorylation then caused a return to the basal level at 20 minutes (Fig 4⇓). Interestingly, TRAP at the highest concentration (10−5 mol/L) was unable to stimulate [3H]thymidine incorporation in these cells (see below), but like thrombin, it stimulated p42MAPK phosphorylation, although with lesser potency (Fig 4⇓).
Thrombin and Cell Proliferation
In cultured SV SMCs, thrombin (1.5×10−8 to 1.5×10−7 mol/L) concentration dependently stimulated [3H]thymidine incorporation (maximum=242±38%, n=4, P<.05), which was much more pronounced than that in the IMA (maximum=151±39, n=4, P<.05) (Fig 5A⇓). In contrast to thrombin, TRAP (10−7 to 10−5 mol/L) had no effects on cell proliferation in either arteries or veins (n=5). The stimulation of [3H]thymidine incorporation by thrombin (3×10−7 mol/L) in SV SMCs was markedly inhibited by 0.5 to 5 U/mL r-hirudin (160±17%, n=6, P<.05 versus thrombin) and 10−6 to 10−5 mol/L PPACK (118±11, n=6, P<.01 versus thrombin) (Fig 5B⇓).
Thrombin Receptor Expression
As demonstrated by RT-PCR, ECs and SMCs from both SV and IMA as well as HUVECs expressed the thrombin receptor (Fig 6⇓). In addition, immunohistological studies with a specific polyclonal antibody against the thrombin receptor in intact IMAs (Fig 7A⇓) and SVs (Fig 7C⇓) showed the presence of thrombin receptor protein expression in endothelium as well as SMCs of both the arteries and veins (brown staining).
This study demonstrates different biological effects of thrombin and thrombin receptor activation on endothelium and smooth muscle of human IMAs and SVs. Although the endothelium and smooth muscle of both arteries and veins expressed the tethered thrombin receptor, activation of the receptor on the endothelium caused endothelium-dependent relaxations (due to release of NO and prostacyclin12 ) in the arteries but not veins. Furthermore, thrombin induced contractions in intact veins but not arteries and induced a much more pronounced proliferation (in cultured SMCs) in the former than the latter. This response in SV SMCs was not mimicked by TRAP, indicating that a mechanism(s) independent of the tethered receptor might exist in venous SMCs that would mediate contraction and proliferation.
Although discovered as a central enzyme in the coagulation cascade, thrombin affects a variety of functions in platelets, monocytes, endothelium, and vascular SMCs. It is likely that not all effects of thrombin occur via the same mechanism. Indeed, in the coagulation cascade, thrombin acts as an enzyme, generating fibrin from fibrinogen.6 These enzymatic effects are crucial for the stabilization of evolving platelet clots. Recently, a tethered thrombin receptor has been cloned.19 During activation, thrombin cleaves the receptor’s N-terminal extracellular domain, generating a new N-terminus that activates the receptor as a tethered ligand. TRAP is a specific tool to activate this tethered receptor. Hence, the results of this study demonstrate that in the IMA thrombin causes endothelium-dependent relaxations that are related to the formation of NO and prostacyclin12 via activation of the tethered thrombin receptor. Indeed, in IMAs the relaxation to thrombin and TRAP were of identical potency. The fact that r-hirudin and PPACK, which bind to and inhibit thrombin’s catalytic epitope, markedly reduced endothelium-dependent relaxations further supports this hypothesis. Finally, RT-PCR and immunohistology demonstrated receptor mRNA and protein in endothelium of IMAs.
In contrast, SVs did not exhibit endothelium-dependent relaxations to thrombin but exhibited contractions. This is not due to the absence of the tethered thrombin receptor, since its presence could be demonstrated in venous endothelium by RT-PCR and immunohistology. Thus, the thrombin receptor in the SV endothelium−although expressed−is not linked to NO synthase or cyclooxygenase.
In quiescent SVs, thrombin caused marked contractions that were similar in preparations with or without endothelium, confirming that thrombin directly contracted SV SMCs. Interestingly, TRAP (at a high concentration that fully mimicked the effects of thrombin in IMAs) caused negligible contractions in SVs. Hence, although the tethered thrombin receptor is expressed in SV SMCs (again confirmed by RT-PCR and immunohistology), contractions to thrombin are not mediated by this receptor. Indeed, in other cells not all responses of thrombin can be explained by activation of the tethered thrombin receptor.11 21 22 24 A second thrombin receptor has been demonstrated in tethered thrombin–receptor gene knockout mice.35 Interestingly, neither thrombin nor TRAP could induce contractions in IMA SMCs even in preparations without endothelium, although these cells express the tethered receptor. Hence, the tethered receptor may exert other unknown functions in IMA SMCs.
Furthermore, we found that thrombin is a more potent stimulus of SMC proliferation of SV than IMA. In venous SMCs, thrombin activated intracellular growth-signaling pathways such as p42MAPK, one of the major mechanisms leading to cell proliferation,27 28 and stimulated DNA synthesis. In contrast, TRAP had no mitogenic effects under these conditions and induced weaker activation of p42MAPK than thrombin. Thus, thrombin stimulates SV SMC proliferation via a mechanism(s) other than activation of the tethered receptor. Alternatively, activation of additional pathway(s), one involving the tethered thrombin receptor and one involving other receptor(s), might be required to exert proliferation. The proliferative as well as contractile effects were inhibited by the thrombin inhibitors r-hirudin and PPACK, which inhibit the catalytic domain of the enzyme. This inhibition suggests that a second receptor that also possesses proteolytic activity35 might be involved.
Similar to the thrombin stimulation of SV SMCs, in IMA SMCs thrombin and TRAP stimulated p42MAPK, demonstrating that thrombin and thrombin receptor activation in arterial SMCs have a functional role in stimulating intracellular signal transduction pathways; however, this leads to much weaker cell proliferation in the artery than the vein. This must be due to the different growth properties of SMCs from IMAs and SVs.5 It is possible that a growth-inhibitory mechanism is dominant in IMA SMCs, which may explain IMA resistance to atherosclerosis as a native vessel and as a graft.1 36
The different responses to thrombin of the IMA and SV may be of importance for their performance as coronary bypass grafts. The observation that SMCs might express more than one thrombin receptor may have important therapeutic implications. Development of a new class of selective thrombin receptor antagonists may have great therapeutic potential.
Selected Abbreviations and Acronyms
|HUVEC||=||human umbilical vein endothelial cell|
|IMA||=||internal mammary artery|
|MAPK||=||mitogen-activated protein kinase|
|PCR||=||polymerase chain reaction|
|SMC||=||smooth muscle cell|
|TRAP||=||thrombin receptor agonist peptide|
This study was supported by the Swiss National Research Foundation (grant No. 32-32541.91/2); the Swiss Cardiology Foundation, Bern; and the Wilhelm Bitter Foundation, Basel, Switzerland. Ton J. Rabelink was sponsored by the Royal Dutch Academy of Sciences.
Presented in part at the 67th Annual Scientific Sessions of the American Heart Association, Dallas, Tex, November 14-17, 1994.
- Received September 4, 1996.
- Revision received November 20, 1996.
- Accepted November 25, 1996.
- Copyright © 1997 by American Heart Association
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