Darcy M. Richardson

Long-Term Vitamin C Treatment Increases Vascular Tetrahydrobiopterin Levels and Nitric Oxide Synthase Activity

Abstract— In cultured endothelial cells, the antioxidant, l-ascorbic acid (vitamin C), increases nitric oxide synthase (NOS) enzyme activity via chemical stabilization of tetrahydrobiopterin. Our objective was to determine the effect of vitamin C on NOS function and tetrahydrobiopterin metabolism in vivo. Twenty-six to twenty-eight weeks of diet supplementation with vitamin C (1%/kg chow) significantly increased circulating levels of vitamin C in wild-type (C57BL/6J) and apolipoprotein E (apoE)–deficient mice. Measurements of NOS enzymatic activity in aortas of apoE-deficient mice indicated a significant increase in total NOS activity. However, this increase was mainly due to high activity of inducible NOS, whereas eNOS activity was reduced. Significantly higher tetrahydrobiopterin levels were detected in aortas of apoE-deficient mice. Long-term treatment with vitamin C restored endothelial NOS activity in aortas of apoE-deficient mice, but did not affect activity of inducible NOS. In addition, 7,8-dihydrobiopterin levels, an oxidized form of tetrahydrobiopterin, were decreased and vascular endothelial function of aortas was significantly improved in apoE-deficient mice. Interestingly, vitamin C also increased tetrahydrobiopterin and NOS activity in aortas of C57BL/6J mice. In contrast, long-term treatment with vitamin E (2000 U/kg chow) did not affect vascular NOS activity or metabolism of tetrahydrobiopterin. In vivo, beneficial effect of vitamin C on vascular endothelial function appears to be mediated in part by protection of tetrahydrobiopterin and restoration of eNOS enzymatic activity.

Coronary vasa vasorum neovascularization precedes epicardial endothelial dysfunction in experimental hypercholesterolemia

OBJECTIVE Experimental hypercholesterolemia is associated with vasa vasorum neovascularization, unknown to occur before or after initial lesion formation. Thus, this study was performed to determine the temporal course of neovascularization of coronary vasa vasorum in relation to endothelial dysfunction, a hallmark of early atherosclerosis. METHODS Female domestic pigs were fed a normal diet (Group 1), a hypercholesterolemic diet for 2 and 4 weeks (Group 2), or a hypercholesterolemic diet for 6 and 12 weeks (Group 3). In vitro analysis of relaxation response to bradykinin served as an index for epicardial endothelial function. Spatial pattern and density of coronary vasa vasorum were assessed by three-dimensional microscopic computed tomography. RESULTS Relaxation response of coronary arteries to bradykinin was normal in both Group 1 (93+/-6%) and Group 2 (89+/-7%) but impaired in Group 3 (71+/-11%; P<0.05 vs. Group 1 and 2). In contrast, density of coronary vasa vasorum was significantly higher in both Group 2 (4.88+/-2.45 per-mm(2)) and Group 3 (4.50+/-1.37 per-mm(2)) compared to Group 1 (2.97+/-1.37 per-mm(2); P<0.05 vs. Group 2 and 3). CONCLUSION This study demonstrates that coronary vasa vasorum neovascularization occurs within the first weeks of experimental hypercholesterolemia and prior to the development of endothelial dysfunction of the host vessel, suggesting a role for vasa vasorum neovascularization in the initial stage of atherosclerotic vascular disease.

Essential Role of Endothelial Nitric Oxide Synthase in Vascular Effects of Erythropoietin

Erythropoietin (EPO) fosters tissue oxygenation by stimulating erythropoiesis. More recently, EPO has been recognized as a tissue-protective cytokine. In this study, we tested the hypothesis that endothelial NO synthase (eNOS) plays a key role in the vascular protective effect of EPO. A murine model of wire-induced injury of carotid artery was used to examine the effect of EPO on endothelial repair and arterial wall architecture. Recombinant human EPO (1000 U/kg, SC, biweekly) was administered for 2 weeks in wild-type and eNOS-deficient mice after which reactivity of isolated carotid arteries was studied in vitro, and the vasculature was histologically assessed. Injured arteries exhibited impairment of endothelium-dependent relaxations to acetylcholine (P<0.05). This was associated with increased medial cross-sectional area (P<0.05). EPO upregulated expression of phosphorylated Ser1177-eNOS and normalized the vasodilator response to acetylcholine (P<0.05). Furthermore, EPO prevented the injury-induced increase in medial cross-sectional area (P<0.05). The vascular protective effects of EPO were abolished in eNOS-deficient mice. Most notably, EPO significantly increased systolic blood pressure and enhanced medial thickening of injured carotid arteries in eNOS-deficient mice (P<0.05). Our results demonstrate that EPO prevents aberrant remodeling of the injured carotid artery. The protective effects of EPO are critically dependent on activation of eNOS.

Chronic antioxidant supplementation attenuates nuclear factor-κB activation and preserves endothelial function in hypercholesterolemic pigs

OBJECTIVE Hypercholesterolemia (HC), a pro-oxidant condition, activates nuclear factor-kappa beta (NF-kappa B) and is associated with coronary endothelial dysfunction. The physiological significance of in vivo chronic antioxidant intervention on HC-induced NF-kappa B activation and coronary endothelial function remains unclear. METHODS Four groups of pigs were studied after 12 weeks of normal diet, normal diet with concomitant antioxidant intervention (100 IU/kg of vitamin E and 1 g of vitamin C daily), 2% HC diet, or HC diet+antioxidant supplementation. NF-kappa B activation and the nitric oxide (NO) pathway were investigated by Western blotting and immunohistochemistry, while oxidative stress was evaluated by coronary artery tissue radical scavenger activity and levels of vitamin E and C. Endothelial function was studied in vitro by coronary vasoreactivity to bradykinin and substance P. RESULTS HC animals had increased activation of NF-kappa B, decreased endothelial NO synthase expression, and decreased radical scavenger system activity, associated with impaired coronary endothelial function. Antioxidant supplementation in HC normalized NF-kappa B activation and NO bioactivity, and preserves coronary endothelial function. CONCLUSIONS This study demonstrates for the first time that in vivo chronic interruption of the endogenous oxidative stress cascade reduces HC-induced NF-kappa B activation and normalizes NO bioactivity in association with preservation of coronary endothelial function. This study suggests a role for increased oxidative stress and NF-kappa B activation in early atherosclerosis.

Insulin and Insulin-like Growth Factor-I Cause Coronary Vasorelaxation In Vitro

Insulin and insulin-like growth factor-I (IGF-I) may play a role in the modulation of coronary artery tone, yet there are few data regarding their vasoactive effects on the coronary vascular bed. We evaluated the vasorelaxation effects of insulin and IGF-I on porcine coronary epicardial vessels in vitro and elucidated possible mechanisms. Porcine epicardial arteries were contracted with 10(-7) mol/L endothelin-1 and relaxed with cumulative concentrations of either insulin or IGF-I (10(-12) to 10(-7) mol/L). The above experiments were repeated in vessels without endothelium. Vessels were also incubated with the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (L-NMMA; 10(-4) mol/L) with and without 10(-3.5) mol/L L-arginine, the potassium channel blocker tetraethylammonium (TEA; 10(-2) mol/L), and the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-alpha]quinoxalin-1-one (ODQ; 10(-5.5) mol/L); vessels were then contracted with endothelin-1 and relaxed with insulin or IGF-I. Insulin and IGF-I were also added after contraction with 60 mmol/L KCl. Insulin and IGF-I caused a similar decrease in coronary epicardial tension after contraction with endothelin-1 (relaxation of 28+/-4% [n=7] and 25+/-3% [n=8] with insulin and IGF-I, respectively; P<0.0001 for both peptides). Removal of the endothelium did not affect these responses. Incubation with L-NMMA, but not ODQ, attenuated the vasorelaxation response to insulin and IGF in vessels without endothelium. L-Arginine did not reverse this effect of L-NMMA. KCl and TEA attenuated the vasorelaxation effect of both insulin and IGF-I. Thus, both insulin and IGF-I caused non-endothelium-dependent coronary vasorelaxation in vitro, probably through a mechanism involving the activation of potassium channels. These findings suggest that insulin and IGF-I participate in the regulation of coronary vasomotor tone.

Coronary Endothelial Function Is Preserved With Chronic Endothelin Receptor Antagonism in Experimental Hypercholesterolemia In Vitro

Hypercholesterolemia is associated with increased circulating and tissue endothelin-1 immunoreactivity, decreased nitric oxide (NO) activity, and altered endothelial function. We tested the hypothesis that chronic endothelin receptor antagonism preserves endothelial function and increases NO in experimental porcine hypercholesterolemia. Pigs were randomized to 3 groups: Group 1, a 2% high-cholesterol (HC) diet alone (n=7); group 2, RO-48-5695, a combined endothelin receptor antagonist, and an HC diet (n=8); and group 3, ABT-627, a selective endothelin-A receptor antagonist, and an HC diet (n=8). Coronary epicardial and arteriolar endothelial function was determined by a dose-response relaxation to bradykinin (10(-11) to 10(-6) mol/L), in all groups and in pigs maintained on a normal diet. Plasma total oxidized products of NO (NO(x)) were determined by chemiluminescence at baseline and after 12 weeks. Bradykinin-stimulated coronary epicardial and arteriolar relaxation in group 1 was attenuated compared with normal-diet controls. This relaxation was normalized with endothelin receptor antagonism. Plasma NO(x) decreased after 12 weeks in group 1 (-74.8+/-5.5%). This decrease was attenuated in the endothelin receptor antagonist groups (group 2, -28.2+/-15.0%; group 3, -38.9+/-20.6%). Chronic endothelin receptor antagonism preserves coronary endothelial function and increases NO in hypercholesterolemia. This study supports a role of endothelin-1 in the regulation of NO activity and suggests a possible therapeutic role for endothelin receptor antagonists in hypercholesterolemia.

Enhanced coronary vasoconstriction to oxidative stress product, 8-epi-prostaglandinF2α, in experimental hypercholesterolemia

OBJECTIVES The F2-isoprostanes are a family of novel prostaglandin isomers and a stable product of in vivo oxidative stress. 8-epi-prostaglandinF2 alpha, a member of this isoprostane family, is a vasoconstrictor and its local release may contribute to the abnormal vasomotor tone associated with hypercholesterolemia. We therefore aimed to outline the role of 8-epi-prostaglandinF2 alpha as a coronary vasoconstrictor in experimental hypercholesterolemia. METHODS AND RESULTS Pigs were randomized to two experimental groups (each n = 9): normal (N) and high cholesterol (HC) diet. To determine the vasoconstrictive effects of 8-epi-prostaglandinF2 alpha in vitro, doses from 10(-9) to 10(-5) M were used to constrict coronary epicardial rings. Plasma total and LDL cholesterol levels were significantly higher in the HC group compared with the N group (P < 0.005) as were plasma 8-epi-prostaglandinF2 alpha levels (P < 0.001). 8-epi-prostaglandinF2 alpha immunoreactivity was present in the vessel wall in both groups. Normal vessels with intact endothelium (n = 8 rings) contracted to 8-epi-prostaglandinF2 alpha (maximal contraction 15.5 +/- 8.74%). In the HC group, rings with intact endothelium had a greater contractile response to 8-epi-prostaglandinF2 alpha compared to normals (72.3 +/- 7.9%; n = 8; P < 0.0001). This was reversed by preincubation with NOR-3, a NO donor (maximal contraction 6.7 +/- 1.56%; n = 5; P < 0.0001). Enhanced contraction in normal vessels occurred with endothelial denudation (98.4 +/- 3.56%; n = 6; P < 0.0001) and with preincubation of the endothelium-intact rings with L-NMMA (N-monomethyl-L-arginine), an NO synthase inhibitor (85.5 +/- 10.3%, n = 6, P < 0.001). The enhanced contraction seen with hypercholesterolemia did not occur with other prostanoid vasoconstrictors. CONCLUSION Experimental hypercholesterolemia leads to a significant increase in 8-epi-prostaglandinF2 alpha levels in addition to enhanced 8-epi-prostaglandinF2 alpha-induced coronary vasoconstriction, in vitro. These findings support a role for the F2-isoprostanes in the regulation of coronary vasomotor tone in pathophysiologic states.

Attenuated In Vitro Coronary Arteriolar Vasorelaxation to Insulin-like Growth Factor I in Experimental Hypercholesterolemia

Insulin and insulin-like growth factor (IGF) 1 affect coronary vasoactivity. Experimental hypercholesterolemia is associated with coronary atherogenesis and altered vasomotor regulation. Because the IGF axis is altered during atherogenesis, we postulated that experimental hypercholesterolemia is associated with an altered coronary vasoactive response to IGF-1 in vitro. Coronary arteries and arterioles from pigs fed either a normal or high-cholesterol diet for 10 weeks were contracted with endothelin-1 and relaxed with cumulative concentrations of insulin or IGF-1 (10(-12) to 10(-7) mol/L). Control arterioles were also incubated with the nitric oxide synthase inhibitor 10(-4) mol/L N(G)-monomethyl-L-arginine (L-NMMA) or the potassium channel blocker 10(-2) mol/L tetraethylammonium (TEA), contracted with endothelin-1, and relaxed with insulin or IGF-1. Experimental hypercholesterolemia (1) increased serum cholesterol (9.5+/-1.0 versus 1.9+/-0.08 mmol/L; P<0.0001), (2) caused coronary arterial and arteriolar endothelial dysfunction in vitro (attenuated vasorelaxation to bradykinin), (3) did not alter the epicardial response to either insulin (P=0.80) or IGF-1 (P=0.12), and (4) significantly attenuated the arteriolar response to IGF-1 (maximal relaxation of 79+/-6% versus 42+/-8%; P=0.01) but not insulin (43+/-6% versus 53+/-7%; P=0.99). Control arteriolar vasorelaxation to IGF-1 was attenuated by both L-NMMA (P<0.001) and TEA (P=0.01), whereas only L-NMMA attenuated insulin (P<0.001). Staining for IGF-1 and IGF binding protein 2 was increased (P<0.05) in arterioles of cholesterol-fed pigs. IGF-1 and insulin are therefore coronary arteriolar vasorelaxants through different mechanisms. Experimental hypercholesterolemia is associated with resistance to the coronary arteriolar vasorelaxing effects of IGF-1 but not insulin, in conjunction with increased ligand and binding-protein expression. The IGF axis may contribute to the altered coronary vasoactivity in hypercholesterolemia.

Insulin and IGF-I attenuate the coronary vasoconstrictor effects of endothelin-1 but not of sarafotoxin 6c

OBJECTIVE To examine the hypothesis that insulin and insulin-like growth factor I (IGF-I) attenuate endothelin-induced contraction of porcine coronary epicardial arteries in vitro. BACKGROUND Endothelin-induced coronary vasoconstriction is mediated by two types of receptors, A (ETA) and B (ETB), resulting in calcium influx. Both insulin and IGF-I attenuate endothelin-induced calcium influx into porcine coronary artery smooth muscle. METHODS Epicardial arteries harvested from juvenile pigs were contracted with cumulative concentrations of endothelin-1 (ETA- and ETB-receptor agonist; 10(-10)-10(-6) M) or of sarafotoxin-6c (ETB-receptor agonist; 10(-11)-10(-7) M). In additional experiments, endothelin-1 or sarafotoxin-6c were added after incubation with 10(-8) M regular insulin or IGF-I. These experiments were repeated in vessels without endothelium. Contraction for each vessel was calculated relative to the response to 60 mM KCl. RESULTS The maximal contractions to endothelin-1 in vessels with and without endothelium were 158 +/- 8 and 200 +/- 21%, respectively (p < 0.05 at 10(-8.5)-10(-6.5) M). Both insulin (at 10(-7)-10(-6) M) and IGF-I (at 10(-6.5)-10(-6) M) attenuated the contraction to endothelin-1 in vessels with intact endothelium, as well as in vessels without endothelium (at 10(-7) and 10(-6) M for insulin and 10(-7.5)-10(-6) M for IGF-I). The maximal contractions to sarafotoxin-6c in vessels with and without endothelium were 54 +/- 13 and 84 +/- 7%, respectively (p < 0.05 at 10(-9), 10(-8.5) and 10(-7) M). Insulin and IGF-I did not affect the response to sarafotoxin-6c in vessels with and without endothelium. CONCLUSION Insulin and IGF-I attenuated ETA-receptor-mediated coronary contraction through an endothelium-independent mechanism. The IGF axis may serve as an endogenous modulator of endothelin-mediated vasoconstriction.