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Ann Thorac Surg 2011;92:1299-1306. doi:10.1016/j.athoracsur.2011.05.114
© 2011 The Society of Thoracic Surgeons
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Original Articles: Adult Cardiac

Endothelium-Dependent Vasoconstriction in Isolated Vessel Grafts: A Novel Mechanism of Vasospasm?

Markus Hoenicka, PhD*, Andreas Keyser, MD, Leopold Rupprecht, MD, Thomas Puehler, MD, Stephan Hirt, MD, Christof Schmid, MD

Department of Cardiothoracic Surgery, University of Regensburg Medical Center, Regensburg, Germany

Accepted for publication May 31, 2011.

* Address correspondence to Dr Hoenicka, Department of Cardiothoracic Surgery, University of Regensburg Medical Center, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany (Email: markus.hoenicka{at}klinik.uni-regensburg.de).


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
Background: YC-1 (3-(5'-hydroxymethyl-2'furyl)-1-benzyl-indazole) is an allosteric activator of soluble guanylyl cyclase (sGC) and a vasodilator. This study describes a paradoxical action of YC-1 in isolated vessels of patients with coronary artery disease (CAD) that appears to trigger an endothelium-dependent vasoconstrictor pathway present in vessels with endothelial dysfunction.

Methods: Effects of YC-1 on the tensions of isolated vessels were investigated in an organ bath. Vasoconstrictors released from the vessels were quantified through enzyme-linked immunosorbent assay.

Results: YC-1 elicited long-lasting constriction in saphenous veins and radial arteries from patients with CAD, but not in human umbilical veins. The half-maximal effective dose was 1.0 µmol/L. Constriction was attenuated by nifedipine (an L-type Ca2+-channel blocker), bosentan (an endothelin [ET]A/ETB inhibitor), BQ-788 (N-[(cis-2,6-Dimethyl-1-piperidinyl)carbonyl]-4-methyl-L-leucyl-1-(methoxycarbonyl)-D-tryptophyl-D-norleucine; an ETB inhibitor), and by denuding, but not by ODQ (1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one; an inhibitor of sGC), BQ-123 (cyclo(-D-Trp-D-Asp-Pro-D-Val-Leu); an ETA inhibitor), or phosphoramidon (an endothelin converting enzyme inhibitor). Indomethacin (an inhibitor of cyclooxygenase-1 and -2) and SQ29,548 ([1S-[1α,2α(Z),3α,4α]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid; a thromboxane receptor antagonist) suppressed YC-1–induced constriction, whereas DFU (5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl)phenyl-2(5H)-furanone; a cyclooxygenase-2 inhibitor) had no effect. Rings of saphenous vein released significantly more endothelin-1 in the presence than in the absence of YC-1.

Conclusions: YC-1–induced vasoconstriction demonstrates the existence of an endothelium-dependent vasoconstrictor pathway in the blood vessels of patients with CAD that to date has been described only in animal models of hypertension. Patients with CAD who have elevated plasma levels of endothelin-1 are thus prone to endothelium-dependent vasoconstriction, which may also play a role in vasospasm in vascular grafts.


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
Coronary vasospasm is one of the major causes of ischemic heart conditions and may lead to stable and unstable angina, myocardial infarction, and sudden death. Endothelial dysfunction, elevated plasma levels of endothelin-1 (ET-1), and reactive oxygen species play a crucial role in the pathogenesis of vasospasm [1].

Vasospasm also affects the patency rates of coronary artery bypass grafts. The risk of vasospasm can be reduced by an appropriate choice of the graft source [2]. Venous grafts are commonly distended, although this may cause structural damage [3]. Arterial grafts are often treated with vasodilators [4] to suppress vasospasm. The nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) system [5] is a target in preventing vasospasm.

YC-1 (3-(5'-hydroxymethyl-2'furyl)-1-benzyl-indazole), an allosteric activator of soluble guanylyl cyclase (sGC), a key enzyme in the guanylyl cyclase system, was found to activate sGC by a mechanism different than that of NO-releasing agents [6]. This discovery initiated the development of vasodilator drugs related to YC-1 for treating arterial and pulmonary hypertension as well as angina pectoris [7]. YC-1 and related compounds activate sGC synergistically with NO and CO [8, 9]. In contrast to organic nitrates, YC-1 and its relatives do not cause tolerance. Consequently, YC-1 and related drugs may be useful agents for preventing vasospasm. Besides its well-known vasodilator action, however, we noticed a paradoxical vasoconstrictor action of YC-1 in segments of vessels from patients with CAD. The study described here investigated key aspects of the mechanism of this vasoconstrictor action and its relationship to endothelial dysfunction.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Comment
 Acknowledgments
 References
 
Study Subjects
Vessel segments were obtained from 157 patients (138 male, 19 female) who underwent elective aortocoronary bypass surgery (Table 1). The patients' mean age was 68.3 ± 7.9 years (range, 48 to 87 years). Risk factors for CAD included hypertension in 132 of the patients, hyperlipidemia in 99, and type 2 diabetes in 49. Only 12 patients were not diagnosed with any of these conditions.


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  		Table 1 Characteristics of Study Patients
 
Harvesting of Blood Vessels
All experiments were approved by the local ethics committee (Ethikkommission der medizinischen Fakultät, Universitätsklinikum Regensburg). Undistended segments of human saphenous vein (HSV) and human radial artery (HRA) were harvested from the patients after their written informed consent was obtained. Human umbilical cords were collected from term pregnancies after written informed consent was obtained from the pregnant women, and umbilical veins (HUV) were dissected from the cords.

Organ-Bath Experiments
The tensions of the vascular specimens were measured in an organ bath as described previously [10, 11]. Cross-sectional rings of the vessels were equilibrated for at least 2 hours, and their resting tensions were adjusted repeatedly to 25 mN. A stable baseline was confirmed by adding KCl (150 mmol/L) to the organ bath, followed by a washout. Receptor-dependent vascular contractions were measured either with norepinephrine or 5-hydroxytryptamine (for HUV).

The actions of vasoconstrictor antagonists were investigated by recording cumulative YC-1 dose–response curves, with a 45-minute incubation period per dose. Antagonists were added 15 minutes before the first dose of YC-1. For recording vasodilator dose–response curves, vessels were constricted with norepinephrine to 80% of their previously established maximum contraction. Serial concentrations of vasodilators were added to all vessel rings except those for the time controls.

Some vessels were denuded of endothelium by rubbing the luminal surface with a wooden toothpick for approximately 60 seconds. Removal of endothelium was ascertained in representative samples by histologic examination and scanning electron microscopy as described previously [10], and YC-1 dose–response curves were constructed. Because denuding may partly damage the vascular smooth-muscle layer, the data on vascular tension from experiments involving denuded vessels were normalized to the vessels' responses to 150 mmol/L KCl.

Measurement of Endothelin Release
Vessel rings were mounted in organ baths. YC-1 was added to each bath, except for the solvent controls which received dimethyl sulfoxide. After 60 minutes, the bath contents were concentrated by approximately 20-fold at 4°C using centrifugal devices (Microsep 1K; Pall; Dreieich, Germany). The concentrates were stored at –80°C and analyzed using an enzyme-linked immunosorbent assay (ELISA) kit (Enzo Life Sciences, Lausen, Switzerland) for endothelin-1.

Data Analysis and Statistical Procedures
Data are presented as mean ± standard deviation with n referring to the number of patients. From 4 to 8 vessel rings were analyzed per subject for each type of experiment. Effects of different doses of a substance were compared with those of the vehicle control through analysis of variance (ANOVA) followed by Dunnett's post-hoc test. Dose–response curves were compared through a two-way repeated measures ANOVA followed by Holm-Sidak post-hoc test. Differences were assumed to be significant for p < 0.05. Half-maximal effective concentrations (EC50) were calculated by the fitting of Hill functions.

Drugs, Chemicals, and Reagents
Sodium nitroprusside (SNP), 1H-(1,2,4)oxadiazole(4,3-a)quinoxalin-1-one (ODQ), NG-nitro-L-arginine-methyl ester (L-NAME), indomethacin, N-(α-rhamnopyranosyloxyhydroxyphosphinyl)-L-leucyl-L-tryptophan (phosphoramidon), BQ-123 (cyclo[D-trp-D-asp-L-pro-D-val-L-leu]), BQ-788 (2,6-Dimethylpiperidinecarbonyl-{gamma}-Methyl-Leu-Nin-(Methoxycarbonyl)-D-Trp-D-Nle, N-[N-[N-[(2,6-Dimethyl-1-piperidinyl)carbonyl]-4-methyl-L-leucyl]-1-(methoxycarbonyl)-D-tryptophyl]-D-norleucine), and SQ29,548 (1S-[1α,2α(Z),3α,4α]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptanoic acid) were obtained from Alexis (Läufelfingen, Switzerland). 5-Hydroxytryptamine (serotonin) and sodium nitroprusside were purchased from Sigma (Taufkirchen, Germany). Norepinephrine was obtained from Aventis (Frankfurt/Main, Germany) and nifedipine was obtained from Bayer (Leverkusen, Germany). The sodium salt of 4-tert-butyl-N-[6-(2-hydroxy-ethoxy)-5-(2-methoxy-phenoxy)-2,2'-bipyrimidin-4-yl]-benzenesulfonamide (bosentan) and 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl)phenyl-2(5H)-furanone (DFU) were generous gifts from Actelion (Allschwil, Switzerland) and from MSD Sharp & Dohme (Haar, Germany), respectively. To exclude artifacts that might be caused by impurities, different batches of YC-1 from two unrelated manufacturers (Alexis and Sigma) were used in the study, and gave identical results.


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Comment
 Acknowledgments
 References
 
YC-1–Induced Constriction of Human Saphenous Vein
Rings of HSV treated with YC-1 (3 µmol/L) developed tonic contractions after a lag phase, and did not return to their resting tensions even after repeated wash cycles (Fig 1 A). Rings precontracted with YC-1 responded to norepinephrine, whereas the NO donor sodium nitroprusside (50 µmol/L) completely relaxed these rings. Contractions of the HSV rings persisted for at least 2 hours (data not shown). Some HSV developed low-frequency oscillations of constriction and relaxation in response to YC-1 (Fig 1B). The median lag between the administration of YC-1 and the onset of contractions was 16.1 minutes (range, 6.0 to 24.8 minutes). The intensity of the contractions showed wide interpatient variability (Fig 1C).


Figure 1
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  		Fig 1. YC-1–induced constriction of rings of human saphenous vein and human radial artery. (A) Representative organ-bath tracing showing the effects on human saphenous vein of 3 µmol/L YC-1 (upper tracing) and dimethylsulfoxide solvent control (lower tracing). Arrows indicate time of administration of test compound; w = washing; NE = serial norepinephrine concentrations from 1 x 10–11 mol/L to 3 x 10–6 mol/L; SNP = sodium nitroprusside at 50 µmol/L added to rings of both human saphenous vein and human radial artery. (B) Effects on rings of human radial artery of same conditions as in A. Note the spontaneous oscillations of constriction and relaxation in this example. (C) Distribution in patient pool of maximum tension induced by 3 µmol/L YC-1. Constrictions induced by both YC-1 and norepinephrine were determined in specimens of human saphenous vein from 134 patients.

 
Differentiation of Relaxing and Constricting Effects of YC-1
Preconstricted vessel rings were relaxed by a series of concentrations of YC-1 (Fig 2). The relaxing effect was noticeable at concentrations of YC-1 as low as 100 nmol/L, although the highest dose did not relax the rings completely. A single dose of the NO donor SNP (10 nmol/L) given before exposure to the series of concentrations of YC-1 sensitized the vessels to YC-1 and increased its relaxant effect. Sodium nitroprusside relaxed the vessels completely, with visible effects beginning at 10 nmol/L.


Figure 2
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  		Fig 2. Organ-bath tracings of YC-1–induced relaxations of human saphenous vein preconstricted with norepinephrine. Arrow indicates addition of 1 x 10–8 mol/L sodium nitroprusside. Vertical marks at bottom of figure indicate sodium nitroprusside dose–response curve from 1 x 10–10 mol/L to 3 x 10–5 mol/L. Crosses indicate YC-1 dose–response curve from 1 x 10–8 mol/L to 3 x 10–5 mol/L. Tracings are representative of 5 independent experiments.

 
To demonstrate vasodilating and vasoconstricting effects of YC-1 on the same vessel, the sGC inhibitor ODQ (10 mmol/L) was added to HSV before recording their YC-1 dose–response curves (Fig 3). In the presence of ODQ, the vessels responded significantly more strongly to doses of YC-1 of 10 µmol/L and 30 µmol/L (p = 0.012, ANOVA, n = 6), indicating that the dilating but not the constricting action of YC-1 can be suppressed by inhibiting the synthesis of cGMP.


Figure 3
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  		Fig 3. Differentiation of vasorelaxant and vasoconstrictive effects of YC-1. Specimens of human saphenous vein were constricted with increasing doses of YC-1 in the absence (filled circles) or presence (open circles) of the soluble guanylyl cyclase inhibitor 1H-(1,2,4)axadiazole(4,3-a)quinoxalin-1-one (ODQ; 10 mmol/L). *Significantly different value for absence and presence of ODQ (p = 0.012, analysis of variance [ANOVA], n = 6).

 
Requirement for Vascular Endothelium
Denuding HSV of their vascular endothelium significantly attenuated but did not completely abolish their responses to YC-1 (p < 0.001, ANOVA, n = 8) (Fig 4).


Figure 4
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  		Fig 4. Dependence of YC-1-induced contractions on endothelium. (A) and (B) Representative hematoxylin-and-eosin-stained cross sections of native and endothelium-denuded human saphenous vein, respectively. (Original magnification in panels A and B, x 10.) (C) YC-1 dose-response curves of native (filled circles) and endothelium-denuded human saphenous vein (open circles). Curves are significantly different (p < 0.001, Analysis of variance [ANOVA], n = 8).

 
Dependence on Calcium Influx
To assess the participation of voltage-gated calcium channels (Cav1.2) in the effect of YC-1, dose–response curves for YC-1 were recorded in the presence and absence of nifedipine (1 µmol/L). In the absence of nifedipine, HSV tensions reached a maximum at concentrations of YC-1 between 3 µmol/L and 10 µmol/L, with an EC50 of 1.00 µmol/L (log[EC50] = –6.00 ± 0.08). Nifedipine completely prevented the YC-1–induced constriction of HSV (p < 0.001, ANOVA, n = 5) (Fig 5). Vasoconstriction induced by YC-1, but not that induced by norepinephrine, was also suppressed in the absence of external Ca2+ (not shown).


Figure 5
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  		Fig 5. Effects of calcium channel, endothelin receptor, endothelin converting enzyme, and thromboxane A2/prostaglandin H2 receptor antagonists on YC-1–induced constriction of human saphenous vein. Cumulative YC-1 dose –response curves (control, filled circles) were constructed after administering nifedipine (1 µmol/L, open circles), bosentan (10 µmol/L, filled triangles), BQ-123 (3 µmol/L, open triangles), BQ-788 (0.5 µmol/L, filled squares), phosphoramidon (10µmol/L, open squares), and SQ29,548 (0.3 µmol/L, filled diamonds). *Significantly different value from control (p < 0.001, analysis of variance[ANOVA], n = 6 or 7).

 
Involvement of Endothelin
The ETA receptor antagonist BQ-123 (3 µmol/L) and the endothelin-converting-enzyme inhibitor phosphoramidon (10 µmol/L) did not affect YC-1 dose–response curves, whereas the mixed ETA/ETB receptor antagonist bosentan (3 µmol/L) and the ETB receptor antagonist BQ-788 (0.5 µmol/L) significantly reduced contractile responses to YC-1 (p < 0.001, ANOVA, n = 6 or 7) (Fig 5), resulting in maximum tensions of 14.6% and 32.0% of those produced by YC-1 without bosentan or BQ-188, respectively.

When rings of HSV were incubated in the presence of different concentrations of YC-1, the accumulation of ET-1 in the baths increased with and increasing YC-1 concentration (p = 0.032, ANOVA, n = 7) (Fig 6).


Figure 6
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  		Fig 6. Endothelin-1 release from rings of human saphenous vein in organ baths after exposure to YC-1 for 60 minutes. The release of endothelin-1 increased significantly with an increasing YC-1 concentration (p = 0.032, analysis of variance [ANOVA], n = 7).

 
Participation of Cyclooxygenase-Derived Prostanoids
Constrictions induced by YC-1 at 3 µmol/L were partly suppressed by 10 µmol/L of the cyclooxygenase (COX)-1/COX-2 inhibitor indomethacin and completely suppressed by 50 µmol/L of indomethacin (p < 0.001, ANOVA n = 4 to 6) (Table 2). In contrast, administration of 1 µmol/L of the COX-2–specific inhibitor DFU did not affect YC-1–induced contractions. The TP receptor antagonist SQ29,548 at 0.3 µmol/L completely suppressed YC-1-induced constrictions (p < 0.001, ANOVA, n = 6) (Fig 5).


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  		Table 2 Effects of Cyclooxygenase Inhibition on YC-1–Induced Contractions of Human Saphenous Vein
 
Evidence of Endothelial Dysfunction
The NO synthase inhibitor L-NAME did not significantly alter the basal tonus of HSV (control, 1.01 ± 2.12 mN vs L-NAME at 100 µmol/L, –0.43 ± 2.17 mN) or the response of HSV to norepinephrine at 1 µmol/L (control, 51.78 ± 15.78 mN vs L-NAME, 56.53 ± 15.42 mN).

Other Types of Vessels
Specimens of HRA were used to determine whether YC-1– induced vasoconstriction is a specific feature of venous vessels of CAD patients. Specimens of HUV were used as readily available human controls. YC-1 was used at 3 µmol/L because it showed no noticeable relaxant effect at this dose (see Fig 3). In this experiment, YC-1 induced the contraction of HRA but not of HUV (p < 0.001, ANOVA, n = 3 to 6) (Table 3).


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  		Table 3 Comparison of Maximum Responses of Vessel Types to YC-1 and Receptor-Mediated Vasoconstrictors
 

   Comment
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
Acknowledgments
 References
 
YC-1 has a variety of molecular targets other than sGC [12–16]. Nevertheless, it was prudent to determine whether sGC is involved in the vasoconstrictor action of YC-1. The sGC inhibitor ODQ [17] permitted distinction of the vasoconstrictor and vasodilator effects of YC-1. The former is sGC-independent, with a calculated EC50 of 1.0 µmol/L, whereas the latter is an sGC-dependent effect that is noticeable only at higher doses of YC-1. This agrees well with published data on the YC-1–induced relaxation of rat and rabbit vessels [18], although the EC50 for the YC-1–induced relaxation of HRA was reported to be approximately 5 nmol/L [19]. This may be due to a higher level of endogenous NO in HRA than in HSV, which would enhance the YC-1–induced stimulation of sGC [9].

The vasorelaxation induced by YC-1 was immediate, whereas YC-1–induced vasoconstriction started after about 15 minutes and developed fully within 60 minutes. These properties suggest that YC-1 operates through a multistep signal-transduction mechanism. Because YC-1–induced vasoconstriction could not be washed out, but was terminated by SNP, the participation of a long-lasting vasoconstrictor in the vasoconstrictive effect of YC-1 appears likely.

Experiments with the denudation of HSV indicated that YC-1–induced vasoconstriction was endothelium-dependent, although a minor contribution of an endothelium-independent mechanism cannot be excluded. Constrictions induced by YC-1, but not those induced by norepinephrine, were inhibited by blocking L-type voltage-gated calcium channels (Cav1.2). Agonist-induced calcium entry into nonexcitable cells depends either on non-voltage–gated calcium channels or on receptor-activated calcium entry [20]. Therefore, YC-1 apparently triggers the release or synthesis of an endothelium-derived vasoconstrictor that depolarizes smooth muscle via Cav1.2 channels.

Endothelin-1 has unique properties that match the characteristics of YC-1–induced contraction. Endothelin-1 is endothelium-derived and elicits contractions of vascular smooth muscle that persist long after this endothelium-derived substance has been removed from the organ bath [21]. Receptors for ETA on smooth muscle induce the phospholipase C pathway without requiring calcium influx [22]. Because YC-1–induced contractions were attenuated by nifedipine but not by antagonizing ET-1A, the direct stimulation of smooth muscle by ET-1 appears unlikely. However, the release of ET-1 increased after the administration of YC-1, and both bosentan (an ETA/ETB inhibitor) and BQ-788 (an ETB inhibitor) attenuated the contractile responses of HSV to YC-1. This indicates that YC-1 triggers endothelial ET-1 release, which acts in a paracrine fashion on ETB receptors. Because the endothelin converting enzyme antagonist phosphoramidon [23] did not affect YC-1–vasoconstriction, ET-1 is apparently released from storage vesicles. Saunders and Scheiner-Bobis [24] reported that the cardiac glycoside ouabain induces the release of ET-1 from endothelial cells within several minutes [25]. The "receptor," if any, that is responsible for transmitting this effect of ouabain has not been identified, but a similar receptor may be involved in YC-1–induced vasoconstriction.

The nonspecific COX inhibitor indomethacin suppressed YC-1–induced vasoconstriction, in contrast to the COX-2–specific inhibitor DFU. Therefore, COX-1 is involved in the synthesis of constricting prostanoids. Prostanoids act on smooth muscle via prostanoid receptors [26]. Further corroborating the notion that vasoconstrictor-induced prostanoid release is essential for the vasoconstrictive action of YC-1 is that this action was completely abolished by the TP receptor antagonist SQ29,548. In rat and canine arteries, thromboxane A2-induced activation of TP receptors requires an influx of external Ca2+, and is therefore affected by the blocking of voltage-gated calcium channels [27–29]. The sensitivity of YC-1–induced vasoconstriction to the calcium-channel blocker nifedipine thus further supports the assumption that TP receptors are involved in the action of YC-1.

Although our data demonstrate the participation of the signaling pathway components described above, further work is needed to unequivocally prove the sequence of events suggested in Figure 7 . However, a strikingly similar mechanism has been suggested to explain the physiological abnormalities in spontaneously hypertensive rats [30], which serve as a model for human hypertension caused by endothelial dysfunction. Rees and colleagues reported the contraction in vitro of healthy vessels from several species of mammals in response to the NOS inhibitor L-NAME [31] as a result of basal release of NO. In contrast, basal HSV tonus in the present study was not affected by L-NAME, and the vessels being tested responded only weakly to endothelium-dependent vasodilators (data not shown), demonstrating endothelial dysfunction. In healthy vessels, the binding of ET to ET-1B receptors on endothelial cells induces vasorelaxation [32], although a constricting mechanism mediated by the ET-1B receptor was demonstrated in rat vessels [33]. This constricting mechanism becomes dominant under pathologic conditions, with ET-1 causing endothelium-dependent vasoconstriction that involves the release of endothelium-derived vasoconstricting factors [34].


Figure 7
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  		Fig 7. Putative mechanism of YC-1–induced vasoconstriction. (AA = arachidonic acid; COX = cyclooxygenase; EC = endothelial cell; ECE = endothelin-converting enzyme; ET-1 = endothelin-1; MLCK = myosin light-chain kinase; PG = prostaglandin; SMC = smooth-muscle cell.)

 
Aging, diabetes, and hypertension contribute to the shift of the endothelium to this constrictive behavior [35, 36, 30]. Both the characteristics and the manifest endothelial dysfunction of our study subjects were in accord with a constrictive endothelial phenotype. Cyclooxygenase-derived prostanoids constitute a major component of endothelium-derived contracting factors (EDCF) [37]. Prostanoids act on smooth muscle via prostanoid receptors [26]. Receptors for thromboxane A2/prostaglandin H2 (TP) have been shown to mediate ET-1–induced vasoconstriction in spontaneously hypertensive rats but not in normotensive control animals [38]. There is in vivo evidence that the same mechanism operates in humans with endothelial dysfunction [39]. The present study is the first to demonstrate this mechanism in isolated human vessels of patients with CAD. Because of the elevated plasma levels of ET-1 in such patients [40], this mechanism may contribute to vasospasm in their coronary arteries and bypass grafts.

The present study has several limitations. Vasoactive medication used by the patients before surgery may have influenced the contractile responses of their vessels, although preliminary analysis did not reveal correlations with any of the patients' characteristics or medications. Specifically, the vessels obtained from the few patients who lacked the common risk factors for atherosclerosis responded to YC-1 as did the vessels of patients with these risk factors. Additionally, the current study was necessarily limited by the specificity of the inhibitors it employed. The release of constricting prostanoids should be further investigated in cell culture models, which was beyond the scope of this initial study.

In summary, this study has shown that the vasodilator YC-1 constricts saphenous veins and radial arteries from patients with severe CAD. The vasoconstriction in both types of vessels is mediated by a paracrine action of endothelium-derived ET-1 that promotes the synthesis of vasoconstrictive prostanoids via COX-1. These prostanoids then constrict vascular smooth muscle via TP receptors. The YC-1–induced constriction of vessels with endothelial dysfunction, and the endothelium-dependent constriction observed in a rat model of hypertension, share major components of their signaling pathways in what may constitute a novel mechanism of vasospasm in coronary arteries and vascular grafts.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
References
 
The authors thank Francesco Santarelli and Katrin Bielenberg (University of Regensburg Medical Center, Department of Cardiothoracic Surgery) for expert technical assistance, and Richard Warth (University of Regensburg, Department of Physiology) for helpful discussions about calcium influx and oscillations.


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 

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