Katušić, Zvonimir S.

In the present study, we used the hph-1 mouse, which displays GTP-cyclohydrolase I (GTPCH I) deficiency, to test the hypothesis that loss of tetrahydrobiopterin (BH4) in conduit and small arteries activates compensatory mechanisms designed to protect vascular wall from oxidative stress induced by uncoupling of endothelial nitric oxide synthase (eNOS). Both GTPCH I activity and BH4levels were reduced in the aortas and small mesenteric arteries of hph-1 mice. However, the BH4-to-7,8-dihydrobiopterin ratio was significantly reduced only in hph-1 aortas. Furthermore, superoxide anion and 3-nitrotyrosine production were significantly enhanced in aortas but not in small mesenteric arteries of hph-1 mice. In contrast to the aorta, protein expression of copper- and zinc-containing superoxide dismutase (CuZnSOD) was significantly increased in small mesenteric arteries of hph-1 mice. Protein expression of catalase was increased in both aortas and small mesenteric arteries of hph-1 mice. Further analysis of endothelial nitric oxide synthase (eNOS)/cyclic guanosine monophosphate (cGMP) signaling demonstrated that protein expression of phosphorylated Ser1177-eNOS as well as basal cGMP levels and hydrogen peroxide was increased in hph-1 aortas. Increased production of hydrogen peroxide in hph-1 mice aortas appears to be the most likely mechanism responsible for phosphorylation of eNOS and elevation of cGMP. In contrast, upregulation of CuZnSOD and catalase in resistance arteries is sufficient to protect vascular tissue from increased production of reactive oxygen species generated by uncoupling of eNOS. The results of our study suggest that anatomical origin determines the ability of vessel wall to cope with oxidative stress induced by uncoupling of eNOS.

The mechanisms underlying the regenerative capacity of endothelial progenitor cells (EPCs) are not fully understood. We hypothesized that biosynthesis of tetrahydrobiopterin is an important mechanism responsible for the stimulatory effects of peroxisome proliferator-activated receptor–δ (PPARδ) activation on regenerative function of human EPCs. Treatment of human EPCs with a selective PPARδ agonist GW501516 for 24 hours increased the levels of mRNA, protein, and enzymatic activity of GTP cyclohydrolase I (GTPCH I), as well as the production of tetrahydrobiopterin. The effects of GW501516 were mediated by suppression of PTEN expression, thereby increasing phosphorylation of AKT. The AKT signaling also mediated GW501516-induced phosphorylation of endothelial NO synthase. In addition, activation of PPARδ significantly enhanced proliferation of EPCs. This effect was abolished by the GTPCH I inhibitor, 2,4-diamino-6-hydroxypyrimidine, or genetic inactivation of GTPCH I with small interfering RNA but not by inhibition of endothelial NO synthase with N G -nitro- l -arginine methyl ester. Supplementation of NO did not reverse 2,4-diamino-6-hydroxypyrimidine-inhibited 5-bromodeoxyuridine incorporation. Furthermore, transplantation of human EPCs stimulated re-endothelialization in a mouse model of carotid artery injury. Pretreatment of EPCs with GW501516 significantly enhanced the ability of transplanted EPCs to repair denuded endothelium. GTPCH I-small interfering RNA transfection significantly inhibited in vivo regenerative capacity of EPCs stimulated with GW501516. Thus, in human EPCs, activation of PPARδ stimulates expression and activity of GTPCH I and biosynthesis of tetrahydrobiopterin via PTEN-AKT signaling pathway. This effect enhances the regenerative function of EPCs.

The study of hemodynamic alterations following the creation of an arteriovenous fistula (AVF) is relevant to vascular adaptive responses and hemodialysis access dysfunction. This study examined such alterations in a murine AVF created by anastomosing the carotid artery to the jugular vein. AVF blood flow was markedly increased due to reduced AVF vascular resistance. Despite such markedly increased basal blood flow, AVF blood flow further increased in response to acetylcholine. This AVF model exhibited increased cardiac output and decreased systemic vascular resistance; the kidney, in contrast, exhibited decreased blood flow and increased vascular resistance. Augmentation in AVF blood flow was attended by increased arterial heme oxygenase-1 (HO-1) mRNA and protein expression, the latter localized to smooth muscle cells of the AVF artery; AVF blood flow was substantially reduced in HO-1−/− mice compared with HO-1+/+ mice. Finally, in a murine model of a representative disease known to exhibit impaired hemodynamic responses (sickle cell disease), the creation of an AVF was attended by decreased AVF flow and impaired AVF function. We conclude that this AVF model exhibits markedly increased AVF blood flow, a vasodilatory reserve capacity, increased cardiac output, decreased renal blood flow, and a dependency on intact hemodynamic responses, in general, and HO-1 expression, in particular, in achieving and maintaining AVF blood flow. We suggest that these findings support the utility of this model in investigating the basis for and the consequences of hemodynamic stress, including shear stress, and the pathobiology of hemodialysis AVF dysfunction.

Background and Purpose —Hypercholesterolemia is associated with abnormal endothelium-dependent vasorelaxation due to decreased nitric oxide bioavailability. Our aim was to examine the effect of adenovirus-mediated gene transfer of endothelial nitric oxide synthase (eNOS) to the hypercholesterolemic rabbit carotid artery in vivo. In addition, we examined whether adenovirus-mediated gene transfer was associated with vascular dysfunction. Methods —Rabbits were fed a 1% cholesterol diet for 4 weeks followed by a 0.5% cholesterol diet for 6 weeks. Vascular reactivity was assessed in nontransduced carotid arteries from chow- and cholesterol-fed animals. In addition, carotid arteries were surgically isolated, and 2 separate doses of adenoviral vectors encoding eNOS or β-galactosidase (AdβGal) on the contralateral side were delivered to the lumen (1×10 10 and 5×10 10 pfu/mL). Results —Abnormal acetylcholine-mediated endothelium-dependent vasorelaxation was detected in the carotid artery from cholesterol-fed animals, whereas responses to calcium ionophore A23187 and diethylamine NONOate were normal. Vascular reactivity was similar in nontransduced and AdβGal-transduced hypercholesterolemic vessels. In vessels transduced with eNOS, transgene expression was demonstrated by immunostaining in both the endothelium and the adventitia and by Western blot analysis. High-dose but not low-dose eNOS gene transfer enhanced endothelium-dependent relaxation in vessels from cholesterol-fed rabbits. Conclusions —Adenovirus-mediated gene transfer of eNOS to carotid arteries of cholesterol-fed animals improves endothelium-dependent relaxation when an optimal viral titer is administered.

Experiments were designed to determine the effect of subarachnoid hemorrhage on endothelium-dependent relaxations in small arteries of the brain stem. A "double-hemorrhage" canine model of the disease was used, and the presence of vasospasm in the basilar artery was confirmed by angiography. Secondary branches of both untreated basilar arteries (inner diameter, 324 +/- 11 microns; n = 12) and arteries exposed to subarachnoid hemorrhage for 7 days (inner diameter, 328 +/- 12 microns; n = 12) were dissected and mounted on glass microcannulas in organ chambers. Changes in the intraluminal diameter of pressurized arteries were measured using a video dimension analyzer. In untreated arteries, 10(-11) to 10(-7) M vasopressin, 10(-10) to 10(-6) M bradykinin, and 10(-9) to 10(-6) M calcium ionophore A23187 caused endothelium-dependent relaxations. At 10(-6) and 3 x 10(-4) M the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) abolished relaxations to vasopressin and produced small but significant rightward shifts of the concentration-response curves to bradykinin and A23187. At 10(-3) M L-arginine prevented the inhibitory effect of L-NAME. Subarachnoid hemorrhage abolished relaxations to vasopressin but did not affect relaxations to bradykinin or A23187. These studies suggest that in small arteries of the brain stem vasopressin causes relaxations by activation of the endothelial L-arginine pathway. This mechanism of relaxation is selectively inhibited by subarachnoid hemorrhage. Preservation of endothelium-dependent relaxations to bradykinin and A23187 is consistent with the concept that small arteries are resistant to vasospasm after subarachnoid hemorrhage.

Background and Purpose—The role of hydrogen peroxide in the regulation of cerebral arterial tone is not completely understood. Previous studies have demonstrated that hydrogen peroxide causes vasodilation of small cerebral arteries. The present study was designed to determine the mechanisms responsible for relaxations of large cerebral arteries to hydrogen peroxide.Methods—Rings of canine middle cerebral arteries without endothelium were suspended for isometric force recording in modified Krebs-Ringer bicarbonate solution bubbled with 94% O2/6% CO2(37°C, pH 7.4). Radioimmunoassay technique was used to determine the levels of cAMP and cGMP.Results—During contraction to UTP (3×10−6or 10−5mol/L), hydrogen peroxide (10−6to 10−4mol/L) caused concentration-dependent relaxations. Catalase (1200 U/mL) abolished the relaxations to hydrogen peroxide. Inhibition of cyclooxygenase by indomethacin (10−5mol/L) significantly reduced relaxations to hydrogen peroxide. In arteries contracted by KCl (20 mmol/L), the relaxations to hydrogen peroxide were significantly reduced. In the presence of a nonselective potassium channel inhibitor, BaCl2(10−4mol/L), a delayed rectifier potassium channel inhibitor, 4-aminopyridine (10−3mol/L), or a calcium-activated potassium channel inhibitor, charybdotoxin (3×10−8mol/L), the relaxations to hydrogen peroxide were also significantly reduced. An ATP-sensitive potassium channel inhibitor, glyburide (5×10−6mol/L), did not affect the relaxations to hydrogen peroxide. Hydrogen peroxide produced concentration-dependent increase in levels of cAMP. Indomethacin (10−5mol/L) inhibited the stimulatory effect of hydrogen peroxide on cAMP production. In contrast, hydrogen peroxide did not affect the levels of cGMP.Conclusions—These results suggest that hydrogen peroxide may cause relaxations of large cerebral arteries in part by activation of arachidonic acid metabolism via cyclooxygenase pathway with subsequent increase in cAMP levels and activation of potassium channels.

Background and PurposeThe mechanisms underlying smooth muscle relaxations of cerebral arteries in response to nitric oxide (NO) and cyclic GMP (cGMP) are still not completely understood. The present study was designed to determine the role of potassium channels in the relaxations to NO donors 3-morpholinosydnonimine (SIN-1) and sodium nitroprusside (SNP), as well as 8-bromo-3′,5′-cGMP (a synthetic analogue of cGMP) and zaprinast (a selective cGMP phosphodiesterase inhibitor).MethodsRings of canine middle cerebral arteries without endothelium were suspended in Krebs-Ringer bicarbonate solution for isometric tension recording. The levels of cGMP were measured by radioimmunoassay. Relaxations to NO donors 8-bromo-cGMP and zaprinast were studied in the presence and in the absence of K+channel blockers charybdotoxin (large-conductance Ca2+-activated K+channels), glyburide (ATP-sensitive K+channels), 4-aminopyridine (delayed rectifier K+channels), and BaCl2(multiple types of K+channels).ResultsConcentration-dependent relaxations caused by NO donors (SIN-1 and SNP) were significantly reduced in arteries treated with BaCl2(3×10−4mol/L) or charybdotoxin (3×10−8mol/L). Relaxations to 8-bromo-cGMP were not affected by the same concentrations of BaCl2and charybdotoxin; however, they were reduced by higher concentrations of BaCl2(3×10−3mol/L) and charybdotoxin (10−7mol/L). Zaprinast-induced relaxations were significantly reduced by BaCl2(3×10−4mol/L) or charybdotoxin (3×10−8mol/L). Glyburide (10−5mol/L) and 4-aminopyridine (10−3mol/L) did not alter the relaxations to SIN-1 or SNP. The production of cGMP stimulated by SIN-1 in the vascular smooth muscle was not affected by BaCl2(3×10−3mol/L) or charybdotoxin (10−7mol/L).ConclusionsThese results indicate that in canine middle cerebral arteries, a significant portion of relaxations to NO liberated from nitrovasodilators is mediated by large-conductance Ca2+-activated K+channels. Other K+channels, sensitive to BaCl2, may also be involved in the mechanism of relaxations induced by NO.

Background and PurposeConcentration of hydrogen ions is an important regulator of cerebral arterial tone under physiological and pathological conditions. Previous studies demonstrated that in cerebral arteries, relaxations to hypercapnia are due to decrease in extracellular pH. The present study was designed to determine the role of potassium channels in mediation of cerebral arterial relaxations induced by extracellular acidosis.MethodsRings of canine basilar arteries without endothelium were suspended for isometric force recording. Acidosis (pH 7.3 to 7.0) was produced by incremental addition of hydrochloric acid (1.0N). The concentration of hydrogen ions was continuously monitored with a pH meter.ResultsDuring contractions to UTP, acidosis (pH 7.3 to 7.0) induced pH-dependent relaxations. These relaxations were abolished in arteries contracted by potassium chloride (20 mmol/L). A nonselective potassium channel inhibitor, BaCl2(10−3and 10−4mol/L), and an ATP-sensitive potassium channel inhibitor, glyburide (5×10−6mol/L), significantly reduced relaxations to acidosis. Furthermore, BaCl2(10−3mol/L) and glyburide (5×10−6mol/L) abolished relaxations to an ATP-sensitive potassium channel opener, cromakalim (10−8to 3×10−5mol/L). However, these potassium channel inhibitors did not affect relaxations to a voltage-dependent calcium channel inhibitor, diltiazem (10−8to 10−4mol/L), and glyburide (5×10−6mol/L) did not alter relaxations to a nitric oxide donor, SIN-1 (10−9to 10−4mol/L). A calcium-activated potassium channel inhibitor, charybdotoxin (10−7mol/L), and a delayed rectifier potassium channel inhibitor, 4-aminopyridine (10−3mol/L), did not affect relaxations to acidosis.ConclusionsThese results suggest that extracellular acidosis causes relaxations of cerebral arteries in part by activation of potassium channels. ATP-sensitive potassium channels appear to contribute to acidosis-induced decrease in cerebral arterial tone.

Endothelin-1 is a very powerful endogenous vasoconstrictor substance produced by endothelial cells. Its long-lasting vasoconstrictor and hypertensive action has been well documented in several species, including humans. It is generally accepted that endothelin-1 may contribute to the pathogenesis of a number of cardiovascular diseases. In the cerebral vasculature, endothelin-1 has been proposed as a key mediator of cerebral vasospasm following subarachnoid hemorrhage. Availability of endothelin-1 antagonist provided a pharmacologic tool to test the role of endothelin in the development of vasospasm. This brief review is focused on the controversial results reported by different groups concerning the possible role of endothelin-1 in narrowing of cerebral arteries exposed to autologous blood.

NG-substituted analogues of L-arginine are potent and selective inhibitors of nitric oxide synthase(s). The present study was designed to determine the effects of these analogues on the vascular smooth muscle of isolated canine basilar arteries. Basilar artery rings without endothelium were suspended for isometric tension recording in Krebs-Ringer bicarbonate solution bubbled with 94% O2-6% CO2 (temperature = 37 degrees C, pH = 7.4). A radioimmunoassay technique was used to determine the levels of guanosine 3',5'-cyclic monophosphate (cyclic GMP). NG-Monomethyl-L-arginine (L-NMMA) caused concentration-dependent contractions, whereas the D-enantiomer and NG-nitro-L-arginine did not. Contractions to L-NMMA were reduced in the presence of L-arginine but not in the presence of D-arginine. Superoxide anions generated by xanthine plus xanthine oxidase in the presence of catalase abolished contractions to L-NMMA but did not affect contractions to the prostaglandin H2/thromboxane A2 agonist U46619. Zaprinast, a selective cyclic GMP phosphodiesterase inhibitor, caused concentration-dependent relaxations. L-NMMA selectively inhibited these relaxations. The inhibitory effect of L-NMMA was reversed in the presence of L-arginine. L-NMMA selectively reduced the basal production of cyclic GMP. These studies suggest that in cerebral arteries, contractions of smooth muscle cells to L-NMMA are mediated by inhibition of nitric oxide synthase with a resultant decrease in the basal production of nitric oxide.