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The vascular endothelium has emerged as a critical determinant of cardiovascular health and disease. Through the release of endothelium derived contracting and relaxing factors, the endothelium modulates vascular tone/reactivity acutely, and in the long term influences the process of vascular remodelling and mitogenesis.1 Hypercholesterolaemia is an important risk factor for coronary endothelial dysfunction. Although a variety of mechanisms have been proposed to link raised cholesterol to diminished endothelial function, recent studies suggest that hypercholesterolaemia may impair vascular function through augmenting the actions of the potent vasoactive peptide endothelin-1.2 Endothelin-1 elicits potent and protracted vasoconstriction; this effect is mediated directly via interaction with endothelin-A and endothelin-B receptors and indirectly via quenching of nitric oxide. Diminished production/availability of nitric oxide is a universal finding in patients with cardiovascular disease.
Tetrahydrobiopterin (BH4) is a cofactor for various enzymatic processes and has been implicated in the pathogenesis of hyperphenylalaninaemia, neurological disorders, Alzheimer's disease, depression, Parkinson's disease, autism, and recently cardiovascular pathology and endothelial dysfunction.3 The balance of published information suggests that in the endothelial cell, BH4 is a critical cofactor for nitric oxide synthase activation, and hence nitric oxide production is dependent upon the presence of adequate amounts of this cofactor. BH4 exerts this action through serving as an electron donor for the hydroxylation of l-arginine. Diminished concentrations of this cofactor, as observed in states of cardiovascular disease, led to an uncoupling of endothelial nitric oxide synthase with diminished nitric oxide and exaggerated superoxide anion production.3 BH4 has emerged as an important target for pharmacological manipulation, and many studies have shown the beneficial effects of BH4 supplementation on endothelial function in vitro and in vivo.
In the present study we hypothesised that acute BH4 treatment may serve to prevent cholesterol induced hyperreactivity to endothelin-1 in vitro in the coronary vasculature. To this aim we studied the effects of the soluble cholesterol derivative, polyoxyethanyl-cholesteryl sebacate (PCS) on endothelin-1 mediated contraction and superoxide production in porcine coronary arteries in the presence and absence of BH4.
Vascular segments of porcine coronary arteries (left anterior descending artery, intact endothelium, n = 7) were studied using standard isometric organ bath procedures as previously described.4 Briefly, following equilibration (resting tension 2 g, Krebs physiological solution), isometric dose response curves to cumulative addition of endothelin-1 (10-10 to 3 × 10-8 M) were constructed in the absence and presence of PCS (30 μg/ml, 10 minutes before endothelin-1 dose, Sigma Inc). These responses were repeated in the presence of BH4 (0.1 mM, 15 minutes, dissolved in 0.5% ascorbic acid). For each concentration, a plateau was obtained before the subsequent dose was added. At the end of the experiment, the tissues were removed, blotted dry, and the cross sectional area of each vascular ring was calculated: cross sectional area (mm2) = weight (mg)/(length (mm) × density (mg/mm3)). The density of the vascular smooth muscle was assumed to be 1.05 mg/mm3. The absolute tension generated was corrected for cross sectional area and expressed as g/mm2.
In a separate study, we examined the effects of endothelin-1, PCS, and BH4 on superoxide anion production in segments of porcine coronary arteries using lucigenin chemiluminescence as described previously.5 Segments of coronary arteries were incubated in Krebs-HEPES buffer for 90 minutes under the following conditions (n = 6 per group): (1) control; (2) control + BH4 (0.1 mM in 0.5% ascorbic acid); (3) control + endothelin-1 (2 nM); (4) control + PCS; (5) control + PCS + endothelin; and (6) control + BH4 + PCS + endothelin-1. BH4 was used as a pretreatment; coronary arteries were incubated with BH4 10 minutes before the additions of other interventions. The segments were transferred to the scintillation vials containing 0.25 mM lucigenin in a final volume of 2 ml.5Data are expressed as counts per second per milligram of tissue (dried weight). Statistical analysis was performed using a one way analysis of variance (ANOVA) followed by a Newman Keul's test. A probability value of p < 0.05 was considered significant between groups.
Endothelin-1 caused a concentration dependent increase in coronary vascular tone (fig 1). Endothelin-1 mediated vasoconstriction was augmented in the presence of the soluble cholesterol derivative. Strikingly, PCS induced coronary hyperreactivity to endothelin-1 was attenuated by co-incubation with BH4 (fig 1). Although BH4 per se caused a significant decrease in endothelin-1 mediated vasoconstriction, the percent reduction of endothelin-1 responses were greater when BH4 was employed in the presence of PCS (40%v 27.9%, p < 0.05).
Dose response curve to endothelin-1 in porcine coronary arteries in the presence and absence of tetrahydrobiopterin (BH4, 0.1 mM) and the soluble cholesterol derivated, polyoxyethanyl-cholesteryl sebacate (PCS, 30 μg/ml). Data are expressed as developed tension (g) corrected for cross sectional area (mm2). In the presence of PCS, endothelin-1 mediated vasoconstriction was enhanced; this response is attenuated by co-incubation with BH4. Although BH4 alone attenuated endothelin-1 responses the degree of attenuation in the presence of PSC was greater (40% v 27.9%, p < 0.05). *p < 0.05 PSC different from control, BH4, and BH4 + PCS. **p < 0.05, BH4 different from control.
Figure 2 depicts the effects of the endothelin-1, PCS, and BH4 on superoxide anion production. Treatment with endothelin-1 alone (2 nM) increased superoxide anion production. This effect was greatly exaggerated in the presence of PCS and attenuated by co-incubation with BH4.
Superoxide anion production expressed as counts per mg tissue in porcine coronary arteries exposed to endothelin-1 (2 nM), PCS (30 μg/ml), and BH4 (0.1 mM). Endothelin-1 increased superoxide anion production by coronary arteries. This response was exaggerated by PCS and attenuated by BH4. Superoxide anion was measured in isolated vascular segments by lucigenin chemiluminescence in the presence of 10 mM diethyldithiocarbamate. *p < 0.05, different from control, BH4, PCS and endothelin (ET) + PSC + BH4. **p < 0.05, different from all groups.
The key observations of the present study are: (1) the coronary vasoconstrictor effects of endothelin-1 are exaggerated by acute in vitro administration of PCS; (2) co-incubation of porcine coronary arteries with the nitric oxide synthase co-factor BH4 attenuates the effects of PCS on endothelin-1 induced vasoreactivity; and (3) the production of superoxide by coronary arteries exposed to PCS + endothelin-1 is greater than each intervention alone. Importantly, this response is attenuated by BH4 supplementation. These data uncover for the first time the functional and acute interaction between cholesterol, endothelin-1, and BH4 in modulating coronary vascular tone.
The synergistic effects of cholesterol on endothelin-1 induced contraction were recently reported in isolated rat aortae.2 Theauthors suggest that this may be mediated through a mitogen activated protein kinase and cyclooxygenase-2 dependent mechanism. The present study extends these observations to the coronary circulation and suggests that diminished production and/or availability of BH4 may be one of the mechanisms mediating the effects of PSC on ET-1 action. Diminished concentrations of BH4 leads to the uncoupling of nitric oxide synthase with the resultant production of superoxide anion and nitric oxide quenching. Supplementation of BH4 may restore endothelial function by preventing the uncoupling of nitric oxide synthase and facilitating the flow of electrons from l-arginine to nitric oxide. Although hypercholesterolaemia and endothelin-1 independently contribute to coronary endothelial dysfunction, we suggest that the synergistic effects of this combination may be ascribed, in part, to diminished availability of BH4. These data are important since patients with raised cholesterol often have coexistent elevations in endothelin-1.6