Basal Nitric Oxide Bioavailability Research Articles

Novel Role of the IGF-1 Receptor in Endothelial Function and Repair

We recently demonstrated that reducing IGF-1 receptor (IGF-1R) numbers in the endothelium enhances nitric oxide (NO) bioavailability and endothelial cell insulin sensitivity. In the present report, we aimed to examine the effect of increasing IGF-1R on endothelial cell function and repair. To examine the effect of increasing IGF-1R in the endothelium, we generated mice overexpressing human IGF-1R in the endothelium (human IGF-1R endothelium-overexpressing mice [hIGFREO]) under direction of the Tie2 promoter enhancer. hIGFREO aorta had reduced basal NO bioavailability (percent constriction to NG-monomethyl-l-arginine [mean (SEM) wild type 106% (30%); hIGFREO 48% (10%)]; P < 0.05). Endothelial cells from hIGFREO had reduced insulin-stimulated endothelial NO synthase activation (mean [SEM] wild type 170% [25%], hIGFREO 58% [3%]; P = 0.04) and insulin-stimulated NO release (mean [SEM] wild type 4,500 AU [1,000], hIGFREO 1,500 AU [700]; P < 0.05). hIGFREO mice had enhanced endothelium regeneration after denuding arterial injury (mean [SEM] percent recovered area, wild type 57% [2%], hIGFREO 47% [5%]; P < 0.05) and enhanced endothelial cell migration in vitro. The IGF-1R, although reducing NO bioavailability, enhances in situ endothelium regeneration. Manipulating IGF-1R in the endothelium may be a useful strategy to treat disorders of vascular growth and repair.

109 Overexpression of endothelial insulin-like growth factor-1 receptors (IGF-1R): a novel role for IGF-1R in endothelial function and repair

IntroductionEndothelium-derived nitric oxide (NO) is a critical regulator of vascular homeostasis, repair and regeneration. We recently demonstrated that reducing insulin-like growth factor-1 receptor (IGF-1R) numbers in the endothelium, thereby decreasing...

Endothelial function in aorta segments of apolipoprotein E-deficient mice before development of atherosclerotic lesions

Acetylcholine (ACh)-induced relaxation declines in apolipoprotein E-deficient (apoE-/-) mouse aortas, but only after atherosclerotic plaque formation. This study investigated intracellular calcium concentrations [Ca2+]i and changes in phenylephrine-induced contractions as index of baseline nitric oxide (NO) bioavailability before plaque development. Isometric contractions of thoracic aorta rings of young (4 months) apoE-/- and C57BL/6J (WT) mice were evoked by phenylephrine (3x10(-9)-3x10(-5) M) in the presence and absence of endothelial cells (ECs) or NO synthase (NOS) inhibitors. [Ca2+]i (Fura-2 AM) and endothelium-dependent relaxation were measured at baseline and after ACh stimulation. Segments of apoE-/- mice were significantly more sensitive and developed more tension than WT segments in response to phenylephrine. The differences disappeared after NOS inhibition or EC removal or upon increasing [Ca2+]i in apoE-/- strips with 10(-6) M cyclopiazonic acid or 10(-7) M Ca2+-ionophore A23187. Expression of endothelial NOS (eNOS) mRNA was similar in apoE-/- and WT aorta segments. Basal [Ca2+]i was significantly lower in apoE-/- than in WT strips. Relaxation by ACh (3x10(-9)-10(-5) M) was time- and dose-dependently related to [Ca2+]i, but neither ACh-induced relaxation nor Ca2+ mobilization were diminished in apoE-/- strips. In conclusion, basal, but not ACh-induced NO bioavailability, was compromised in lesion-free aorta of apoE-/- mice. Decreased basal NO bioavailability was not related to lower eNOS expression, but most likely related to lower basal [Ca2+]i. These findings further point to important differences between basal and stimulated eNOS activity.

Estrogen Treatment Enhances Nitric Oxide Bioavailability in Normotensive but not Hypertensive Rats

The effects of estrogen on endothelial function remain controversial. Endothelial function is perturbed in hypertension. We aimed to determine whether pre-existing hypertension can modify endothelial-dependent responses to estrogen. We compared the effects of estrogen replacement on endothelial function in healthy female adult Wistar Kyoto (WKY) rats and stroke-prone spontaneously hypertensive rats (SHRSP). Basal and carbachol-stimulated nitric oxide (NO) bioavailability were studied in carotid artery rings in ovariectomized animals treated with estrogen or placebo for 2 weeks in vivo, or after 1 h of incubation in vitro. Basal NO bioavailability was defined as the increase in pressor responses in phenylephrine in the presence of NO synthase blockade. Superoxide (O(2)(-)) levels in aortas were measured by lucigenin chemiluminescence and endothelial NO synthase (eNOS) protein levels by Western blotting. Basal NO bioavailability was increased in WKY treated with estrogen for 2 weeks compared to placebo. In contrast, no change in NO bioavailability was observed in SHRSP. The O(2)(-) levels were higher in SHRSP than in WKY but unaffected by estrogen treatment in either strain. In WKY, but not in SHRSP, estrogen caused upregulation of eNOS. Similarly in vitro exposure to estrogen increased NO bioavailability in WKY but had no effect in SHRSP. In WKY, co-exposure to estrogen and LY294002, a PI3 kinase inhibitor, abrogated the effect of estrogen. The inability of estrogen to improve endothelial function in SHRSP may relate to a defect in eNOS activation pathways in this hypertensive rat strain.

Reduced basal nitric oxide bioavailability and platelet activation in young spontaneously hypertensive rats

Reduced basal nitric oxide bioavailability and platelet activation in young spontaneously hypertensive rats

391. Targeted Delivery of Adenovirus Expressing eNOS to the Endothelium In Vivo Lowers Blood Pressure in Hypertensive Rats

Top of pageAbstract Background: Vascular dysfunction associated with oxidative stress is a key feature of human essential hypertension. The stroke prone spontaneously hypertensive rat (SHRSP) is a model of this condition. We have shown that local in vivo application of adenovirus encoding for either endothelial nitric oxide synthase (eNOS) or extracellular superoxide dismutase (SOD3) improves impaired nitric oxide (NO) bioavailability and endothelial function in the carotid artery of the SHRSP (1,2). The present study sought to determine the effects of systemic administration of these vectors on blood pressure in the SHRSP. Since adenovirus is sequestered and degraded by the liver we also evaluated specific targeting of vectors to the vascular endothelium, using a bispecific antibody 3. Methods: Systolic blood pressure (SBP) was recorded by radiotelemetry in 10 to 11 week-old male SHRSP for a total of 8 weeks. Four hours after infusion of 3×1011 particles of null adenovirus (to saturate hepatic Kupffer cells), rats received a single infusion of 1×1010 particles of recombinant adenoviral vectors ('RAd'), or vehicle, encoding for eNOS, SOD3 or EGFP via the femoral vein (n = 5 per group). Two further groups (n = 5) received RAdeNOS or RAdEGFP pre-incubated with a bispecific antibody (Fab9B9) which binds to both the adenoviral coat and angiotensin converting enzyme. Telemetry data was analysed using a Linear Mixed Effects Model. Results: Biodistribution studies using Lightcycler real-time PCR revealed that the vast majority of non-targeted adenoviral particles accumulated in liver, with lower levels in spleen, lung, aorta and carotid artery. Transgene expression, determined by immunohistochemistry, paralleled this pattern. Non-targeted over-expression of extracellular SOD or eNOS had no effect on SBP. However, endothelial targeting increased vector accumulation in the lung, and pulmonary eNOS immunoreactivity was increased in rats which had received Fab9B9-RAdeNOS but not Fab9B9-RAdEGFP. Functional studies found that these changes were accompanied by a significant increase in basal NO bioavailability in small pulmonary arteries. Moreover, in contrast to non-targeted vectors, Fab9B9-RAdeNOS caused a significant and sustained reduction in SBP relative to the Fab9B9-RAdEGFP control (see Figure 1). Conclusion: This study shows for the first time that specific vascular targeting of adenoviral-mediated gene delivery can modify systemic cardiovascular phenotype.

The HMG-CoA reductase inhibitor cerivastatin enhances the nitric oxide bioavailability of the endothelium.

The aim of this study was to investigate whether the HMG-CoA reductase inhibitor cerivastatin alters the nitric oxide (NO) bioavailability of porcine aortic endothelial cell cultures and of native porcine coronary endothelium, after short-term (minutes) and long-term (24-hour) treatment with cerivastatin (electrochemical NO sensor). NO-synthase expression (Western blot, ELISA) and activity (3H-arginine assay) after cerivastatin treatment were determined. Furthermore, the authors investigated whether cerivastatin modulates an angiotensin II (10 micromol/L; 4 hours) induced reactive oxygen species (ROS) release from intact vessels (lucigenin-enhanced chemiluminescence-assay). Acute addition of cerivastatin induced a concentration-dependent NO release from endothelial cell cultures that could be blocked by the NO-synthase inhibitor N-monomethyl-arginine. A long-term incubation with cerivastatin also resulted in a concentration-dependent, significantly enhanced basal NO bioavailability (approximately 3-fold increase at 10 nmol/L cerivastatin) that could be partly reversed by a coincubation with mevalonate. No enhanced endothelial NO-synthase expression or increased NO-synthase activity was detected after long-term treatment with cerivastatin (24 hours). However, cerivastatin induced a significant concentration-dependent inhibition of the angiotensin II-induced ROS release from native endothelial cells of porcine coronary arteries. Therefore, there seems to be an acute and a long-term effect of cerivastatin that results in enhanced endothelial NO bioavailability.

Effects of Peroxynitrite on the Reactivity of Diabetic Rat Aorta

Endogenous nitric oxide (NO) reacts with superoxide to form peroxynitrite, which is capable of either oxidizing or nitrating various biological substrates. We compared the vasodilatory effect of exogenous peroxynitrite with the effects of decomposed peroxynitrite or sodium nitrite in precontracted aorta isolated from streptozotocin-induced diabetic and age-matched control rats. Peroxynitrite (10 nmol/l to 300 µmol/l) produced a concentration-dependent relaxation in aortic rings with or without endothelium. Relaxation was also observed with a higher concentration of its decomposition product or sodium nitrite, although these relaxations were considerably slower and with reduced sensitivity. Endothelium-containing rings were less sensitive to the vasorelaxant effect of peroxynitrite than the endothelium-denuded rings in control (pD₂ was 5.19 ± 0.06 in rings with endothelium and 5.86 ± 0.03 in rings without endothelium, p < 0.01) but not in diabetic aorta (pD₂ was 5.97 ± 0.05 in rings with endothelium and 6.12 ± 0.06 in rings without endothelium, p > 0.05). The maximum relaxation to peroxynitrite also increased in diabetics, but did not change by removal of the endothelium either in diabetic or control rings. Diabetes did not alter the relaxations elicited by both decomposed peroxynitrite and sodium nitrite. Peroxynitrite-induced relaxation was not inhibited by diethylenetriaminepentaacetic acid, an inhibitor of hydroxyl radical formation. Pretreatment with peroxynitrite (1 µmol/l, 15 min) significantly suppressed the phenylephrine-induced tone and acetylcholine-stimulated endothelium-dependent relaxation, both effects were more pronounced in diabetic than in control aorta. The increased responsiveness of diabetic vessels to exogenous peroxynitrite seems to be related to depressed basal NO bioavailability and may be considered as a compensatory way against activated contractile mechanisms of diabetic vascular smooth muscle.