A change in the redox environment and thromboxane A2 production precede endothelial dysfunction in mice

We reported that the endothelial dysfunction that develops with age was associated with a proinflammatory phenotype. In this study, we hypothesized that an increased production of proinflammatory cyclooxygenase (COX) products occurs before endothelial dysfunction. Dilations to acetylcholine (ACh) were recorded from pressurized renal arteries isolated from 3- and 6-mo-old C57Bl/6 male mice treated or not with the polyphenol catechin (30 mg x kg(-1) x day(-1)) in drinking water for 3 mo. Release of thromboxane (TX) B(2), the metabolite of TXA(2), was measured by using immunoenzymatic assays, and free radical production was measured by using the fluorescent dye CM-H(2)DCFDA. Endothelial nitric oxide synthase (eNOS) and COX-1/2 mRNA expression were quantified by quantitative PCR. N(G)-nitro-L-arginine (L-NNA) reduced (P < 0.05) ACh-induced dilation in vessels isolated from 3- and 6-mo-old mice. In the presence of L-NNA, indomethacin normalized (P < 0.05) the dilation in vessels from 6-mo-old mice only. SQ-29548 (PGH(2)/TXA(2) receptor antagonist) and furegrelate (TXA(2) synthase inhibitor), in the presence of L-NNA, also improved (P < 0.05) dilation. L-NNA increased TXA(2) release and free radical-associated fluorescence, the latter being prevented by SQ-29548. In vessels from 6-mo-old mice treated with catechin for 3 mo, L-NNA-dependent reduction in ACh-mediated dilation was insensitive to indomethacin, whereas TXA(2) release and free radical-associated fluorescence were prevented. eNOS mRNA expression was significantly increased by catechin treatment. Our results suggest that an augmented production of TXA(2) and the associated change in redox regulation precede the development of the endothelial dysfunction.     

Catechin treatment improves cerebrovascular flow-mediated dilation and learning abilities in atherosclerotic mice

Severe dyslipidemia and the associated oxidative stress could accelerate the age-related decline in cerebrovascular endothelial function and cerebral blood flow (CBF), leading to neuronal loss and impaired learning abilities. We hypothesized that a chronic treatment with the polyphenol catechin would prevent endothelial dysfunction, maintain CBF responses, and protect learning abilities in atherosclerotic (ATX) mice. We treated ATX (C57Bl/6-LDLR(-/-)hApoB(+/+); 3 mo old) mice with catechin (30 mg · kg(-1) · day(-1)) for 3 mo, and C57Bl/6 [wild type (WT), 3 and 6 mo old] mice were used as controls. ACh- and flow-mediated dilations (FMD) were recorded in pressurized cerebral arteries. Basal CBF and increases in CBF induced by whisker stimulation were measured by optical coherence tomography and Doppler, respectively. Learning capacities were evaluated with the Morris water maze test. Compared with 6-mo-old WT mice, cerebral arteries from 6-mo-old ATX mice displayed a higher myogenic tone, lower responses to ACh and FMD, and were insensitive to NOS inhibition (P < 0.05), suggesting endothelial dysfunction. Basal and increases in CBF were lower in 6-mo-old ATX than WT mice (P < 0.05). A decline in the learning capabilities was also observed in ATX mice (P < 0.05). Catechin 1) reduced cerebral superoxide staining (P < 0.05) in ATX mice, 2) restored endothelial function by reducing myogenic tone, improving ACh- and FMD and restoring the sensitivity to nitric oxide synthase inhibition (P < 0.05), 3) increased the changes in CBF during stimulation but not basal CBF, and 4) prevented the decline in learning abilities (P < 0.05). In conclusion, catechin treatment of ATX mice prevents cerebrovascular dysfunctions and the associated decline in learning capacities.     

Chronic high blood flow potentiates shear stress-induced release of NO in arteries of aged rats

Aging impairs shear-stress-dependent dilation of arteries via increased superoxide production, decreased SOD activity, and decreased activation of endothelial nitric oxide (NO) synthase (eNOS). In the present study, we investigated whether chronic increases in shear stress, elicited by increases in blood flow, would improve vascular endothelial function of aged rats. To this end, second-order mesenteric arteries of young (6 mo) and aged (24 mo) male Fischer-344 rats were selectively ligated for 3 wk to elevate blood flow in a first-order artery [high blood flow (HF)]. An in vitro study was then conducted on first-order arteries with HF and normal blood flow (NF) to assess shear stress (1, 10, and 20 dyn/cm(2))-induced release of NO into the perfusate. In HF arteries of both age groups, shear stress-induced NO production increased significantly. In 24-mo-old rats, the reduced shear stress-induced NO production in NF arteries was normalized by HF to a level similar to that in NF arteries of 6-mo-old rats. The increased NO production in HF arteries of 24-mo-old rats was associated with increased shear stress-induced dilation, expression of eNOS protein, and shear stress-induced eNOS phosphorylation. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, reduced shear stress-induced eNOS phosphorylation and vasodilation. Superoxide production decreased significantly in HF compared with NF arteries in 24-mo-old rats. The decreased superoxide production was associated with significant increases in CuZn-SOD and extracellular SOD protein expressions and total SOD activity. These results suggest that stimulation with chronic HF restores shear-stress-induced activation of eNOS and antioxidant ability in aged arteries.     

Loss of alpha2B-adrenoceptors increases magnitude of hypertension following nitric oxide synthase inhibition

Vascular alpha(2B)-adrenoceptors (alpha(2B)-AR) may mediate vasoconstriction and contribute to the development of hypertension. Therefore, we hypothesized that blood pressure would not increase as much in mice with mutated alpha(2B)-AR as in wild-type (WT) mice following nitric oxide (NO) synthase (NOS) inhibition with N(omega)-nitro-l-arginine (l-NNA, 250 mg/l in drinking water). Mean arterial pressure (MAP) was recorded in heterozygous (HET) alpha(2B)-AR knockout mice and WT littermates using telemetry devices for 7 control and 14 l-NNA treatment days. MAP in HET mice was increased significantly on treatment days 1 and 4 to 14, whereas MAP did not change in WT mice (days 0 and 14 = 113 +/- 3 and 114 +/- 4 mmHg in WT, 108 +/- 0.3 and 135 +/- 13 mmHg in HET, P < 0.05). MAP was significantly higher in HET than in WT mice days 10 through 14 (P < 0.05). Thus blood pressure increased more rather than less in mice with decreased alpha(2B)-AR expression. We therefore examined constrictor responses to phenylephrine (PE, 10(-9) to 10(-4) M) with and without NOS inhibition to determine basal NO contributions to arterial tone. In small pressurized mesenteric arteries (inner diameter = 177 +/- 5 microm), PE constriction was decreased in untreated HET arteries compared with WT (P < 0.05). l-NNA (100 microM) augmented PE constriction more in HET arteries than in WT arteries, and responses were not different between groups in the presence of l-NNA. Acetylcholine dilated preconstricted arteries from HET mice more than arteries from WT mice. Endothelial NOS expression was increased in HET compared with WT mesenteric arteries by Western analysis. Griess assay showed increased NO(x) concentrations in HET plasma compared with those in WT plasma. These data demonstrate that diminished alpha(2B)-AR expression increases the dependence of arterial pressure and vascular tone on NO production and that vascular alpha(2B)-AR either directly or indirectly regulates vascular endothelial NOS function.     

COX-2 contributes to the maintenance of flow-induced dilation in arterioles of eNOS-knockout mice

Our previous studies demonstrated that, in gracilis muscle arterioles of male mice deficient in the gene for endothelial nitric oxide synthase (eNOS), flow-induced dilation (FID) is mediated by endothelial PGs. Thus the present study aimed to identify the specific isoform of cyclooxygenase (COX) responsible for the compensatory mediation of FID in arterioles of eNOS-knockout (KO) mice. Experiments were conducted on gracilis muscle arterioles of male eNOS-KO and wild-type (WT) mice. Basal tone and magnitude of FID of arterioles were comparable in the two strains of mice. A role for COX isoforms in the mediation of the responses was assessed by use of valeryl salicylate (3 mM) and NS-398 (10 microM), inhibitors of COX-1 and COX-2, respectively. In eNOS-KO arterioles, valeryl salicylate or NS-398 alone inhibited FID (at maximal flow rate) by approximately 51% and approximately 58%, respectively. Administration of both inhibitors eliminated the dilation. In WT arterioles, inhibition of COX-2 did not significantly affect FID, whereas inhibition of COX-1 decreased the dilation by approximately 57%. The residual portion of the response was abolished by additional administration of Nomega-nitro-L-arginine methyl ester. Western blot analysis indicated a comparable content of COX-1 protein in arterioles of WT and eNOS-KO mice. COX-2 protein, which was not detectable in arterioles of WT mice, was strongly expressed in arterioles of eNOS-KO mice, together with an upregulation of COX-2 gene expression. Immunohistochemical staining confirmed the presence of COX-2 in the endothelium of eNOS-KO arterioles. In conclusion, COX-2-derived PGs are the mediators responsible for maintenance of FID in arterioles of eNOS-deficient mice.     

Regulation of nitric oxide-dependent vasodilation in coronary arteries of estrogen receptor-alpha-deficient mice

Estrogen has been shown to increase endothelium-dependent vasodilation and expression of endothelial nitric oxide (NO) synthase (eNOS); however, the role of estrogen receptors in mediating estrogen effects on endothelial function remains to be elucidated. The purpose of this study was to test the hypothesis that estrogen modulates NO-dependent vasodilation of coronary arteries through its action on estrogen receptor-alpha (ER-alpha) to increase protein levels of eNOS and Cu/Zn superoxide dismutase (SOD-1). Vasodilation to acetylcholine (ACh) and sodium nitroprusside was assessed in isolated coronary arteries from intact and ovariectomized female wild-type (WT) and ER-alpha knockout (ERalphaKO) mice. Protein levels for eNOS and SOD-1 were also evaluated. Vasodilation to ACh was not significantly altered in ERalphaKO mice compared with WT mice. Ovariectomy reduced responsiveness to ACh in ERalphaKO mice but not WT mice. Responses to sodium nitroprusside were not altered by disruption of ER-alpha or by ovariectomy. Supplementation with estrogen restored ACh-induced vasodilation in ovariectomized ERalphaKO mice. eNOS protein was reduced in ERalphaKO mice compared with WT mice. Ovariectomy caused a further reduction in eNOS protein in ERalphaKO mice, but this reduction was reversed by estrogen treatment. SOD-1 protein levels were increased by disruption of ER-alpha. Ovariectomy reduced SOD-1 protein in ERalphaKO mice, but this reduction was partially reversed by estrogen replacement. These results suggest that estrogen modulation of eNOS protein content is mediated in part through ER-alpha. NO-dependent responses are preserved in ERalphaKO mice, possibly through increased SOD-1 expression and enhanced bioavailability of NO.     

COX-2 is involved in vascular oxidative stress and endothelial dysfunction of renal interlobar arteries from obese Zucker rats

Obesity is related to vascular dysfunction through inflammation and oxidative stress and it has been identified as a risk factor for chronic renal disease. In the present study, we assessed the specific relationships among reactive oxygen species (ROS), cyclooxygenase 2 (COX-2), and endothelial dysfunction in renal interlobar arteries from a genetic model of obesity/insulin resistance, the obese Zucker rats (OZR). Relaxations to acetylcholine (ACh) were significantly reduced in renal arteries from OZR compared to their counterpart, the lean Zucker rat (LZR), suggesting endothelial dysfunction. Blockade of COX with indomethacin and with the selective blocker of COX-2 restored the relaxations to ACh in obese rats. Selective blockade of the TXA₂/PGH₂ (TP) receptor enhanced ACh relaxations only in OZR, while inhibition of the prostacyclin (PGI₂) receptor (IP) enhanced basal tone and inhibited ACh vasodilator responses only in LZR. Basal production of superoxide was increased in arteries of OZR and involved NADPH and xanthine oxidase activation and NOS uncoupling. Under conditions of NOS blockade, ACh induced vasoconstriction and increased ROS generation that were augmented in arteries from OZR and blunted by COX-2 inhibition and by the ROS scavenger tempol. Hydrogen peroxide (H₂O₂) evoked both endothelium- and vascular smooth muscle (VSM)-dependent contractions, as well as ROS generation that was reduced by COX-2 inhibition. In addition, COX-2 expression was enhanced in both VSM and endothelium of renal arteries from OZR. These results suggest that increased COX-2-dependent vasoconstriction contributes to renal endothelial dysfunction through enhanced (ROS) generation in obesity. COX-2 activity is in turn upregulated by ROS.     

Short-term increases in intraluminal pressure reverse age-related decrements in endothelium-dependent dilation in soleus muscle feed arteries.

We tested the hypothesis that short-term increases in intraluminal pressure improve endothelium-dependent dilation and increase endothelial nitric oxide (NO) synthase (eNOS) expression in senescent soleus muscle feed arteries (SFA). SFA isolated from young (4 mo) and old (24 mo) Fischer 344 rats were cannulated and pressurized at 90 (p90) or 130 (p130) cmH(2)O for 4 h. At the end of the 4-h protocol, pressure in p130 SFA was lowered to 90 cmH(2)O for examination of endothelium-dependent (flow- or ACh-induced) vasodilation. Flow- and ACh-induced dilations were blunted in old p90 SFA relative to young p90 SFA. Pretreatment with increased pressure (p130) improved flow- and ACh-induced dilations in old SFA, such that vasodilator responses were similar to those in young SFA. In the presence of N(omega)-nitro-l-arginine (l-NNA) or l-NNA + indomethacin (Indo), flow-induced dilation was inhibited in old p130 SFA, such that the response was not greater than the response in old p90 SFA. In old p130 SFA, ACh-induced dilation was inhibited by l-NNA + Indo (not l-NNA alone). In a separate experiment, SFA were pressurized at 70, 90, 110, or 130 cmH(2)O for 4 h, and eNOS mRNA and protein content were assessed. Increased pressure induced eNOS mRNA expression in young (not old) SFA. eNOS protein content was not altered in young or old SFA. These results indicate that short-term increases in intraluminal pressure improve endothelium-dependent dilation in senescent SFA, in part by enhancing NO bioavailability; however, the beneficial effect was not associated with increased eNOS expression.     

In eNOS knockout mice skeletal muscle arteriolar dilation to acetylcholine is mediated by EDHF

The mechanisms that account for acetylcholine (ACh)-induced responses of skeletal muscle arterioles of mice lacking endothelial nitric oxide (NO) synthase (eNOS-KO) were investigated. Isolated, cannulated, and pressurized arterioles of gracilis muscle from male eNOS-KO (74.1 +/- 2.3 microm) and wild-type (WT, 87.2 +/- 2.1 microm) mice developed spontaneous tone accounting for 63 and 61% of their passive diameter (116.8 +/- 3.4 vs. 143.2 +/- 2.8 microm, respectively) and dilated dose-dependently to ACh (10(-9)-10(-7) M). These dilations were significantly smaller in vessels of eNOS-KO compared with WT mice (29.2 +/- 2.0 microm vs. 46.3 +/- 2.1 microm, at maximum concentration) but responses to the NO donor, sodium nitrite (NaNO(2), 10(-6)-3 x 10(-5) M), were comparable in the vessels of the two strains. N(G)-nitro-L-arginine (L-NNA, 10(-4) M), an inhibitor of eNOS, inhibited ACh-induced dilations by 60-90% in arterioles of WT mice but did not affect responses in those of eNOS-KO mice. In arterioles of eNOS-KO mice, dilations to ACh were not affected by indomethacin but were essentially abolished by inhibitors of cytochrome P-450, clotrimazole (CTZ, 2 x 10(-6) M) or miconazole (MCZ, 2 x 10(-6) M), as well as by either high K(+) (40 mM) or iberiotoxin [10(-7) M, a blocker of Ca(2+)-dependent K(+) channels (K(Ca) channels)]. On the other hand, in WT arterioles CTZ or MCZ inhibited ACh-induced dilations only by approximately 10% and only in the presence of L-NNA. These results indicate that in arterioles of eNOS-KO mice, endothelium-derived hyperpolarizing factor (EDHF), synthesized via cytochrome P-450, accounts entirely for the mediation of ACh-induced dilation via an increase in K(Ca)-channel activity. In contrast, in arterioles of WT mice, endothelium-derived NO predominantly mediates ACh-induced dilation in which participation of EDHF becomes apparent only after inhibition of NO synthesis.     

AGE/RAGE produces endothelial dysfunction in coronary arterioles in type 2 diabetic mice

We hypothesized that impaired nitric oxide (NO)-dependent dilation (endothelial dysfunction) in type 2 diabetes results, in part, from elevated production of superoxide (O(2)(*-)) induced by the interaction of advanced glycation end products (AGE)/receptor for AGE (RAGE) and TNF-alpha signaling. We assessed the role of AGE/RAGE and TNF-alpha signaling in endothelial dysfunction in type 2 diabetic (Lepr(db)) mice by evaluation of endothelial function in isolated coronary resistance vessels of normal control (nondiabetic, m Lepr(db)) and diabetic mice. Although dilation of vessels to the endothelium-independent vasodilator sodium nitroprusside (SNP) was not different between diabetic and control mice, dilation to the endothelium-dependent agonist acetylcholine (ACh) was reduced in diabetic vs. control mice. The activation of RAGE with RAGE agonist S100b eliminated SNP-potentiated dilation to ACh in Lepr(db) mice. Administration of a soluble form of RAGE (sRAGE) partially restored dilation in diabetic mice but did not affect dilation in control mice. The expression of RAGE in coronary arterioles was markedly increased in diabetic vs. control mice. We also observed in diabetic mice that augmented RAGE signaling augmented expression of TNF-alpha, because this increase was attenuated by sRAGE or NF-kappaB inhibitor MG132. Protein and mRNA expression of NAD(P)H oxidase subunits including NOX-2, p22(phox), and p40(phox) increased in diabetic compared with control mice. sRAGE significantly inhibited the expression of NAD(P)H oxidase in diabetic mice. These results indicate that AGE/RAGE signaling plays a pivotal role in regulating the production/expression of TNF-alpha, oxidative stress, and endothelial dysfunction in type 2 diabetes.     

Deletion of Protein Tyrosine Phosphatase 1B (PTP1B) Enhances Endothelial Cyclooxygenase 2 Expression and Protects Mice from Type 1 Diabetes-Induced Endothelial Dysfunction

Protein tyrosine phosphatase 1B (PTP1B) dephosphorylates receptors tyrosine kinase and acts as a molecular brake on insulin signaling pathway. Conditions of metabolic dysfunction increase PTP1B, when deletion of PTP1B protects against metabolic disorders by increasing insulin signaling. Although vascular insulin signaling contributes to the control of glucose disposal, little is known regarding the direct role of PTP1B in the control of endothelial function. We hypothesized that metabolic dysfunctions increase PTP1B expression in endothelial cells and that PTP1B deletion prevents endothelial dysfunction in situation of diminished insulin secretion. Type I diabetes (T1DM) was induced in wild-type (WT) and PTP1B-deficient mice (KO) with streptozotocin (STZ) injection. After 28 days of T1DM, KO mice exhibited a similar reduction in body weight and plasma insulin levels and a comparable increase in glycemia (WT: 384±20 vs. Ko: 432±29 mg/dL), cholesterol and triglycerides, as WT mice. T1DM increased PTP1B expression and impaired endothelial NO-dependent relaxation, in mouse aorta. PTP1B deletion did not affect baseline endothelial function, but preserved endothelium-dependent relaxation, in T1DM mice. NO synthase inhibition with L-NAME abolished endothelial relaxation in control and T1DM WT mice, whereas L-NAME and the cyclooxygenases inhibitor indomethacin were required to abolish endothelium relaxation in T1DM KO mice. PTP1B deletion increased COX-2 expression and PGI₂ levels, in mouse aorta and plasma respectively, in T1DM mice. In parallel, simulation of diabetic conditions increased PTP1B expression and knockdown of PTP1B increased COX-2 but not COX-1 expression, in primary human aortic endothelial cells. Taken together these data indicate that deletion of PTP1B protected endothelial function by compensating the reduction in NO bioavailability by increasing COX-2-mediated release of the vasodilator prostanoid PGI₂, in T1DM mice.     

Accelerated endothelial dysfunction in mild prediabetic insulin resistance: the early role of reactive oxygen species

Insulin resistance is well established as an independent risk factor for the development of type 2 diabetes and cardiovascular atherosclerosis. Most studies have examined atherogenesis in models of severe insulin resistance or diabetes. However, by the time of diagnosis, individuals with type 2 diabetes already demonstrate a significant atheroma burden. Furthermore, recent studies suggest that, even in adolescence, insulin resistance is a progressive disorder that increases cardiovascular risk. In the present report, we studied early mechanisms of reduction in the bioavailability of the antiatheroscerotic molecule nitric oxide (NO) in very mild insulin resistance. Mice with haploinsufficiency for the insulin receptor (IRKO) are a model of mild insulin resistance with preserved glycemic control. We previously demonstrated that 2-mo-old (Young) IRKO mice have preserved vasorelaxation responses to ACh. This remained the case at 4 mo of age. However, by 6 mo, despite no significant deterioration in glucose homeostasis (Adult), IRKO mice had marked blunting of ACh-mediated vasorelaxation [IRKO maximum contraction response (E(max)) 66 +/- 5% vs. wild type 87 +/- 4%, P < 0.01]. Despite the endothelial dysfunction demonstrated, aortic endothelial nitric oxide synthase (eNOS) mRNA levels were similar in Adult IRKO and wild-type mice, and, interestingly, aortic eNOS protein levels were increased, suggesting a compensatory upregulation in the IRKO. We then examined the potential role of reactive oxygen species in mediating early endothelial dysfunction. The superoxide dismutase mimetic Mn(III)tetrakis(1-methyl-4-pyridyl) porphyrin pentachloride (MnTMPyP) restored ACh relaxation responses in the Adult IRKO (E(max) to ACh with MnTMPyP 85 +/- 5%). Dihydroethidium fluorescence of aortas and isolated coronary microvascular endothelial cells confirmed a substantial increase in endothelium-derived reactive oxygen species in IRKO mice. These data demonstrate that mild insulin resistance is a potent substrate for accelerated endothelial dysfunction and support a role for endothelial cell superoxide production as a mechanism underlying the early reduction in NO bioavailability.     

Transcriptional basis for exercise limitation in male eNOS-knockout mice with age: heart failure and the fetal phenotype

Endothelium-derived nitric oxide (NO) is pivotal in regulating mitochondrial O(2) consumption (Vo(2)) and glucose uptake in mice. The aim of this study was to investigate the mechanism of age- and genotype-related exercise limitation in male endothelial NO synthase (eNOS)-knockout (KO, n = 16) and wild-type (WT, n = 19) mice. Treadmill testing was performed at 12, 14, 16, 18, and 21 mo of age. Vo(2), CO(2) production, respiratory exchange ratio, and maximal running distance were determined during treadmill running. There were good linear correlations for increase of speed with increase of Vo(2). The difference between KO and WT mice was not significant at 12 mo but was significant at 18 mo. Linear regression showed that KO mice consumed more O(2) at the same absolute and relative workloads, suggesting that Vo(2) was not inhibited by NO in KO mice. KO mice performed 30-50% less work than WT mice at each age (work = vertical distance x weight). In contrast to WT mice, the work performed by KO mice significantly decreased from 17 +/- 1.4 m.kg at 12 mo to 9.4 +/- 1.7 m.kg at 21 mo. Running distance was significantly decreased from 334 +/- 27 m at 12 mo to 178 +/- 38 m at 21 mo, and maximal Vo(2), CO(2) production, and respiratory exchange ratio per work unit were significantly higher in KO than in WT mice. Gene arrays showed evidence of a fetal phenotype in KO mice at 21 mo. In conclusion, age- and genotype-related exercise limitations in maximal work performed and maximal running distance in male eNOS-KO mice indicated that fetal phenotype and age were related to onset of heart failure.     

Interaction between nitric oxide and prostanoids in arterioles of rat cremaster muscle in vivo

We studied in vivo interactions of nitric oxide (NO), oxidative stress, and prostanoids derived from the cyclooxygenase pathway in the arterioles studied by intravital microscopy in peripheral muscle. Topical administration of NO synthase (NOS) inhibitor Nomega-nitro-l-arginine (l-NNA) or cyclooxygenase inhibitor mefenamic acid (MA) alone leads to vasoconstriction. We found that l-NNA after MA induced an additional constriction, whereas MA after l-NNA induced a relative dilation. Therefore, an additional constriction was found when MA was administered after l-NNA in the presence of the thromboxane A2 synthase-PGH2 (TP) receptor antagonist SQ-29548. We also found a relative dilation when the TP receptor antagonist was administered after NOS inhibition by l-NNA. In the presence of superoxide dismutase and catalase, l-NNA-induced vasoconstriction is reduced, and the dilation observed after addition of MA in presence of the reactive oxygen species is no longer present. Taken together, these results showed that NO inhibition induced a shift in the synthesis or in the effects of cyclooxygenase products, in favor of constrictor prostanoids. This effect of NO inhibition disappears when reactive oxygen species are scavenged by superoxide dismutase and catalase.     

Agonist-dependent variablity of contributions of nitric oxide and prostaglandins in human skeletal muscle.

The relative contributions of endothelium-dependent dilators [nitric oxide (NO), prostaglandins (PGs), and endothelium-derived hyperpolarizing factor (EDHF)] in human limbs are poorly understood. We tested the hypothesis that relative contributions of NO and PGs differ between endothelial agonists acetylcholine (ACh; 1, 2, and 4 microg.dl(-1).min(-1)) and bradykinin (BK; 6.25, 25, and 50 ng.dl(-1).min(-1)). We measured forearm blood flow (FBF) using venous occlusion plethysmography in 50 healthy volunteers (27 +/- 1 yr) in response to brachial artery infusion of ACh or BK in the absence and presence of inhibitors of NO synthase [NOS; with NG-monomethyl-L-arginine (L-NMMA)] and cyclooxygenase (COX; with ketorolac). Furthermore, we tested the idea that the NOS + COX-independent dilation (in the presence of L-NMMA + ketorolac, presumably EDHF) could be inhibited by exogenous NO administration, as reported in animal studies. FBF increased approximately 10-fold in the ACh control; L-NMMA reduced baseline FBF and ACh dilation, whereas addition of ketorolac had no further effect. Ketorolac alone did not alter ACh dilation, but addition of L-NMMA reduced ACh dilation significantly. For BK infusion, FBF increased approximately 10-fold in the control condition; L-NMMA tended to reduce BK dilation (P < 0.1), and addition of ketorolac significantly reduced BK dilation. Similar to ACh, ketorolac alone did not alter BK dilation, but addition of L-NMMA reduced BK dilation. To test the idea that NO can inhibit the NOS + COX-independent portion of dilation, we infused a dose of sodium nitroprusside (NO-clamp technique) during ACh or BK that restored the reduction in baseline blood flow due to L-NMMA. Regardless of treatment order, the NO clamp restored baseline FBF but did not reduce the NOS + COX-independent dilation to ACh or BK. We conclude that the contribution of NO and PGs differs between ACh and BK, with ACh being more dependent on NO and BK being mostly dependent on a NOS + COX-independent mechanism (EDHF) in healthy young adults. The NOS + COX-independent dilation does not appear sensitive to feedback inhibition from NO in the human forearm.     

Exacerbation of endothelial dysfunction during the progression of diabetes: role of oxidative stress

To test the deterioration of endothelial function during the progression of diabetes, shear stress-induced dilation (SSID; 10, 20, and 40 dyn/cm(2)) was determined in isolated mesenteric arteries (80-120 μm in diameter) of 6-wk (6W), 3-mo (3M), and 9-mo (9M)-old male db/db mice and their wild-type (WT) controls. Nitric oxide (NO)-mediated SSID was comparable in 6W WT and db/db mice, but the dilation was significantly reduced in 3M db/db mice and declined further in 9M db/db mice. Vascular superoxide production was progressively increased in 3M and 9M db/db mice, associated with an increased expression of NADPH oxidase. Inhibition of NADPH oxidase significantly improved NO-mediated SSID in arteries of 3M, but not in 9M, db/db mice. Although endothelial nitric oxide synthase (eNOS) expression was comparable in all groups, a progressive reduction in shear stress-induced eNOS phosphorylation existed in vessels of 3M and 9M db/db mice. Moreover, inducible NOS (iNOS) that was not detected in WT, nor in 6W and 3M db/db mice, was expressed in vessels of 9M db/db mice. A significantly increased expression of nitrotyrosine in total protein and immunoprecipitated eNOS was also found in vessels of 9M db/db mice. Thus, impaired NO bioavailability plays an essential role in the endothelial dysfunction of diabetic mice, which becomes aggravated when endothelial nitrosative stress is further activated via perhaps, an additional iNOS-mediated pathway during the progression of diabetes.     

Celecoxib reduces hepatic vascular resistance in portal hypertension by amelioration of endothelial oxidative stress

The balance between endothelial nitric oxide (NO) synthase (eNOS) activation and production of reactive oxygen species (ROS) is very important for NO homeostasis in liver sinusoidal endothelial cells (LSECs). Overexpression of cyclooxygenase-2 (COX-2), a major intravascular source of ROS production, has been observed in LSECs of cirrhotic liver. However, the links between low NO bioavailability and COX-2 overexpression in LSECs are unknown. This study has confirmed the link between low NO bioavailability and COX-2 overexpression by COX-2-dependent PGE2-EP2-ERK1/2-NOX1/NOX4 signalling pathway in LSECs in vivo and in vitro. In addition, the regulation of COX-2-independent LKB1-AMPK-NRF2-HO-1 signalling pathway on NO homeostasis in LSECs was also elucidated. The combinative effects of celecoxib on diminishment of ROS via COX-2-dependent and COX-2-independent signalling pathways greatly decreased NO scavenging. As a result, LSECs capillarisation was reduced, and endothelial dysfunction was corrected. Furthermore, portal hypertension of cirrhotic liver was ameliorated with substantial decreasing hepatic vascular resistance and great increase of portal blood flow. With the advance understanding of the mechanisms of LSECs protection, celecoxib may serve as a potential therapeutic candidate for patients with cirrhotic portal hypertension.     

Gender and aging in a transgenic mouse model of hypertrophic cardiomyopathy

Mutations in the cardiac myosin heavy chain (MHC) can cause familial hypertrophic cardiomyopathy (FHC). A transgenic mouse model has been developed in which a missense (R403Q) allele and an actin-binding deletion in the alpha-MHC are expressed in the heart. We used an isovolumic left heart preparation to study the contractile characteristics of hearts from transgenic (TG) mice and their wild-type (WT) littermates. Both male and female TG mice developed left ventricular (LV) hypertrophy at 4 mo of age. LV hypertrophy was accompanied by LV diastolic dysfunction, but LV systolic function was normal and supranormal in the young TG females and males, respectively. At 10 mo of age, the females continued to present with LV concentric hypertrophy, whereas the males began to display LV dilation. In female TG mice at 10 mo of age, impaired LV diastolic function persisted without evidence of systolic dysfunction. In contrast, in 10-mo-old male TG mice, LV diastolic function worsened and systolic performance was impaired. Diminished coronary flow was observed in both 10-mo-old TG groups. These types of changes may contribute to the functional decompensation typically seen in hypertrophic cardiomyopathy. Collectively, these results further underscore the potential utility of this transgenic mouse model in elucidating pathogenesis of FHC.     

NAD(P)H oxidase-generated superoxide anion accounts for reduced control of myocardial O2 consumption by NO in old Fischer 344 rats

We investigated the role of nitric oxide (NO) in the control of myocardial O2 consumption in Fischer 344 rats. In Fischer rats at 4, 14, and 23 mo of age, we examined cardiac function using echocardiography, the regulation of cardiac O2 consumption in vitro, endothelial NO synthase (eNOS) protein levels, and potential mechanisms that regulate superoxide. Aging was associated with a reduced ejection fraction [from 75 +/- 2% at 4 mo to 66 +/- 3% (P < 0.05) at 23 mo] and an increased cardiac diastolic volume [from 0.60 +/- 0.04 to 1.00 +/- 0.10 ml (P < 0.01)] and heart weight (from 0.70 +/- 0.02 to 0.90 +/- 0.02 g). The NO-mediated control of cardiac O2 consumption by bradykinin or enalaprilat was not different between 4 mo (36 +/- 2 or 34 +/- 3%) and 14 mo (29 +/- 1 or 25 +/- 3%) but markedly (P < 0.05) reduced in 23-mo-old Fischer rats (15 +/- 3 or 7 +/- 2%). The response to the NO donor S-nitroso-N-acetyl penicillamine was not different across groups (35%, 35%, and 44%). Interestingly, the eNOS protein level was not different at 4, 14, and 23 mo. The addition of tempol (1 mmol/l) to the tissue bath eliminated the depression in the control of cardiac O2 consumption by bradykinin (25 +/- 3%) or enalaprilat (28 +/- 3%) in 23-mo-old Fischer rats. We next examined the levels of enzymes involved in the production and breakdown of superoxide. The expression of Mn SOD, Cu/Zn SOD, extracellular SOD, and p67phox, however, did not differ between 4- and 23-mo-old rats. Importantly, there was a marked increase in gp91phox, and apocynin restored the defect in NO-dependent control of cardiac O2 consumption at 23 mo to that seen in 4-mo-old rats, identifying the role of NADPH oxidase. Thus increased biological activity of superoxide and not decreases in the enzyme that produces NO are responsible for the altered control of cardiac O2 consumption by NO in 23-mo-old Fischer rats. Increased oxidant stress in aging, by decreasing NO bioavailability, may contribute not only to changes in myocardial function but also to altered regulation of vascular tone and the progression of cardiac or vascular disease.