Akt1 is critical for acute inflammation and histamine-mediated vascular leakage

Akt1 is implicated in cell metabolism, survival migration, and gene expression; however, little is known about the role of specific Akt isoforms during inflammation in vivo. Thus, we directly explored the roles of the isoforms Akt1 and Akt2 in acute inflammation models by using mice deficient in either Akt1 or Akt2. Akt1^−/− mice showed a markedly reduced edema versus Akt2^−/− and WT controls, and the reduced inflammation was associated with a dramatic decrease in neutrophil and monocyte infiltration. The loss of Akt1 did not affect leukocyte functions in vitro, and bone marrow transplant experiments suggest that host Akt1 regulates leukocyte emigration into inflamed tissues. Moreover, carrageenan-induced edema and the direct propermeability actions of bradykinin and histamine were reduced dramatically in Akt1^−/− versus WT mice. These findings are supported by in vitro experiments showing that Akt1 deficiency or blockade of nitric oxide synthase markedly reduces histamine-stimulated changes in transendothelial electrical resistance of microvascular endothelial cells. Collectively, these results suggest that Akt1 is necessary for acute inflammation and exerts its actions primarily via regulation of vascular permeability, leading to edema and leukocyte extravasation.     

Endothelial mechanotransduction, nitric oxide and vascular inflammation

Numerous aspects of vascular homeostasis are modulated by nitric oxide and reactive oxygen species (ROS). The production of these is dramatically influenced by mechanical forces imposed on the endothelium and vascular smooth muscle. In this review, we will discuss the effects of mechanical forces on the expression of the endothelial cell nitric oxide synthase, production of ROS and modulation of endothelial cell glutathione. We will also review data that exercise training in vivo has a similar effect as laminar shear on endothelial function and discuss the clinical relevance of these basic findings.     

内皮型一氧化氮合酶基因多态性与急性心肌梗死的相关性探讨

目的　探讨内皮型一氧化氮合酶 (eNOS)基因多态性与急性心肌梗死 (AMI)的相关性。方法　依据eNOS基因外显子 7G894T位点设计引物 ,通过巢式聚合酶链反应 (PCR)扩增目的片段 ,限制性内切酶消化目的片段 ,琼脂糖凝胶电泳 ,紫外透射分析仪检测 ,计数 10 7例AMI病人及 81例健康者基因型及突变基因频率 ,通过χ2 检验有无统计学意义。结果　eNOS基因外显子 7的 894位点有 3种基因型 :GG、GT、TT。AMI组 10 7例中2 5例发生G894T突变 ,纯合子TT 9例 ,杂合子GT 16例。对照组 81例中 13例发生G894T突变 ,均为杂合子。两组等位基因纯合子突变具有非常显著统计学意义 ,x2 =5 4 2 9,P <0 0 5 ,两组等位基因总突变率 (纯合子 +杂合子 )无明显统计学意义 ,x2 =1 5 2 9,P >0 0 5。结论　eNOS基因 894位点TT型突变与AMI发病密切相关 ,是AMI发病的危险因子     

Endothelial nitric oxide synthase activation and nitric oxide function: new light through old windows

The principle mechanisms operating at the level of endothelial nitric oxide synthase (eNOS) itself to control its activity are phosphorylation, the auto-regulatory properties of the protein itself, and Ca(2)(+)/calmodulin binding. It is now clear that activation of eNOS is greatest when phosphorylation of certain serine and threonine residues is accompanied by elevation of cytosolic [Ca2+](i). While eNOS also contains an autoinhibitory loop, Rafikov et al. (2011) present the evidence for a newly identified 'flexible arm' that operates in response to redox state. Boeldt et al. (2011) also review the evidence that changes in the nature of endothelial Ca(2)(+) signaling itself in different physiologic states can extend both the amplitude and duration of NO output, and a failure to change these responses in pregnancy is associated with preeclampsia. The change in Ca(2)(+) signaling is mediated through altering capacitative entry mechanisms inherent in the cell, and so many agonist responses using this mechanism are altered. The term 'adaptive cell signaling' is also introduced for the first time to describe this phenomenon. Finally NO is classically regarded as a regulator of vascular function, but NO has other actions. One proposed role is regulation of steroid biosynthesis but the physiologic relevance was unclear. Ducsay & Myers (2011) now present new evidence that NO may provide the adrenal with a mechanism to regulate cortisol output according to exposure to hypoxia. One thing all three of these reviews show is that even after several decades of study into NO biosynthesis and function, there are clearly still many things left to discover.     

Endothelial nitric oxide synthase in vascular disease: from marvel to menace

Nitric oxide (NO*) is an important protective molecule in the vasculature, and endothelial NO* synthase (eNOS) is responsible for most of the vascular NO* produced. A functional eNOS oxidizes its substrate L-arginine to L-citrulline and NO*. This normal function of eNOS requires dimerization of the enzyme, the presence of the substrate L-arginine, and the essential cofactor (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4), one of the most potent naturally occurring reducing agents. Cardiovascular risk factors such as hypertension, hypercholesterolemia, diabetes mellitus, or chronic smoking stimulate the production of reactive oxygen species in the vascular wall. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases represent major sources of this reactive oxygen species and have been found upregulated and activated in animal models of hypertension, diabetes, and sedentary lifestyle and in patients with cardiovascular risk factors. Superoxide (O2*-) reacts avidly with vascular NO* to form peroxynitrite (ONOO-). The cofactor BH4 is highly sensitive to oxidation by ONOO-. Diminished levels of BH4 promote O2*- production by eNOS (referred to as eNOS uncoupling). This transformation of eNOS from a protective enzyme to a contributor to oxidative stress has been observed in several in vitro models, in animal models of cardiovascular diseases, and in patients with cardiovascular risk factors. In many cases, supplementation with BH4 has been shown to correct eNOS dysfunction in animal models and patients. In addition, folic acid and infusions of vitamin C are able to restore eNOS functionality, most probably by enhancing BH4 levels as well.     

Endothelial nitric oxide synthase is critical for ischemic remodeling, mural cell recruitment, and blood flow reserve

The genetic loss of endothelial-derived nitric oxide synthase (eNOS) in mice impairs vascular endothelial growth factor (VEGF) and ischemia-initiated blood flow recovery resulting in critical limb ischemia. This result may occur through impaired arteriogenesis, angiogenesis, or mobilization of stem and progenitor cells. Here, we show that after ischemic challenge, eNOS knockout mice [eNOS (-/-)] have defects in arteriogenesis and functional blood flow reserve after muscle stimulation and pericyte recruitment, but no impairment in endothelial progenitor cell recruitment. More importantly, the defects in blood flow recovery, clinical manifestations of ischemia, ischemic reserve capacity, and pericyte recruitment into the growing neovasculature can be rescued by local intramuscular delivery of an adenovirus encoding a constitutively active allele of eNOS, eNOS S1179D, but not a control virus. Collectively, our data suggest that endogenous eNOS-derived NO exerts direct effects in preserving blood flow, thereby promoting arteriogenesis, angiogenesis, and mural cell recruitment to immature angiogenic sprouts.     

Endothelial dihydrofolate reductase: critical for nitric oxide bioavailability and role in angiotensin II uncoupling of endothelial nitric oxide synthase

Recent studies demonstrate that oxidative inactivation of tetrahydrobiopterin (H4B) may cause uncoupling of endothelial nitric oxide synthase (eNOS) to produce superoxide (O2*-). H4B was found recyclable from its oxidized form by dihydrofolate reductase (DHFR) in several cell types. Functionality of the endothelial DHFR, however, remains completely unknown. Here we present findings that specific inhibition of endothelial DHFR by RNA interference markedly reduced endothelial H4B and nitric oxide (NO.) bioavailability. Furthermore, angiotensin II (100 nmol/liter for 24 h) caused a H4B deficiency that was mediated by H2O2-dependent down-regulation of DHFR. This response was associated with a significant increase in endothelial O2*- production, which was abolished by eNOS inhibitor N-nitro-L-arginine-methyl ester or H2O2 scavenger polyethylene glycol-conjugated catalase, strongly suggesting H2O2-dependent eNOS uncoupling. Rapid and transient activation of endothelial NAD(P)H oxidases was responsible for the initial burst production of O2* (Rac1 inhibitor NSC 23766 but not an N-nitro-L-arginine-methyl ester-attenuated ESR O2*- signal at 30 min) in response to angiotensin II, preceding a second peak in O2*- production at 24 h that predominantly depended on uncoupled eNOS. Overexpression of DHFR restored NO. production and diminished eNOS production of O2*- in angiotensin II-stimulated cells. In conclusion, these data represent evidence that DHFR is critical for H4B and NO. bioavailability in the endothelium. Endothelial NAD(P)H oxidase-derived H2O2 down-regulates DHFR expression in response to angiotensin II, resulting in H4B deficiency and uncoupling of eNOS. This signaling cascade may represent a universal mechanism underlying eNOS dysfunction under pathophysiological conditions associated with oxidant stress.     

Endothelial nitric oxide synthase regulation is altered in pancreas from cirrhotic rats

AIM: To determine whether biliary cirrhosis could induce pancreatic dysfunction such as modifications in endothelial nitric oxide synthase(eNOS) expression and whether the regulation of eNOS could be altered by the regulatory proteins caveolin and heat shock protein 90 (Hsp90), as well as by the modifications of calmodulin binding to eNOS. METHODS: Immunoprecipitations and Western blotting analysis were performed in pancreas isolated from sham and cirrhotic rats. RESULTS: Pancreatic injury was minor in cirrhotic rats but eNOS expression importantly decreased with the length (and the severity) of the disease. Because co-immunoprecipitation of eNOS with both Hsp90 and caveolin similarly decreased in cirrhotic rats, eNOS activity was not modified by this mechanism. In contrast, cirrhosis decreased the calmodulin binding to eNOS with a concomitant decrease in eNOS activity. CONCLUSION: In biliary cirrhosis, pancreatic injury is minor but the pancreatic nitric oxide (NO) production is significantly decreased by two mechanisms: a decreased expression of the enzyme and a decreased binding of calmodulin to eNOS.     

Endothelial nitric oxide synthase polymorphisms and hypertension

The human endothelial nitric oxide synthase (eNOS) gene is highly polymorphic. Evidence for the involvement of eNOS single nucleotide polymorphisms in the development of essential hypertension is limited, though the eNOS Glu298Asp polymorphism appears to influence the blood pressure response to exercise. This variant also influences endothelial function, with its effects becoming manifest during the adaptive vascular changes of pregnancy. Carriers of eNOS Asp298 may be at risk of developing pre-eclampsia. Molecular studies have indicated that intact eNOS Asp298 has equivalent enzymatic activity to eNOS Glu298, but undergoes selective proteolysis in native cells and tissues such that the steady state level of active eNOS may be reduced in carriers of this allele. Carriers of eNOS Asp298, particularly if exposed to adverse environmental infuences on endothelial function, may be at increased risk of developing atherosclerosis and cerebrovascular disease.     

Endothelial nitric oxide synthase and nitric oxide regulate endothelial tissue factor expression in vivo in the sickle transgenic mouse

Activation of the coagulation system is a characteristic feature of sickle cell anemia, which also includes clinical thrombosis. The sickle transgenic mouse abnormally expresses tissue factor (TF) on the pulmonary vein endothelium. Knowing that this aberrancy is stimulated by inflammation, we sought to determine whether nitric oxide (NO) contributes to regulation of endothelial TF expression in the sickle mouse model. We used the NY1DD sickle mouse, which exhibits a low‐TF to high‐TF phenotype switch on exposure to hypoxia/reoxygenation. Manipulations of NO biology, such as breathing NO or addition of arginine or L ‐NAME (N‐nitro‐L ‐arginine‐methyl‐ester) to the diet, caused significant modulations of TF expression. This was also seen in hBERK1 sickle mice, which have a different genetic background and already have high‐TF even at ambient air. Study of NY1DD animals bred to overexpress endothelial nitric oxide synthase (eNOS; eNOS‐Tg) or to have an eNOS knockout state (one eNOS^?/? animal and several eNOS^+/? animals) demonstrated that eNOS modulates endothelial TF expression in vivo by down‐regulating it. Thus, the biodeficiency of NO characteristic of patients with sickle cell anemia may heighten risk for activation of the coagulation system. Am. J. Hematol., 2010. © 2009 Wiley‐Liss, Inc.     

Endothelial nitric oxide synthase and endothelial dysfunction

Nitric oxide (NO) regulates vascular tone and local blood flow, platelet aggregation and adhesion, and leukocyte-endothelial cell interactions. Abnormalities in NO production by the vascular endothelium result in endothelial dysfunction, which occurs in hypertension, diabetes, aging, and as a prelude to atherosclerosis. The common feature of endothelial dysfunction is a decrease in the amount of bioavailable NO. In this article, the physiologic roles of NO and the mechanisms of endothelial dysfunction are reviewed. Regulation of endothelial NO synthase (eNOS) activity by fatty acid modifications, intracellular localization, interactions with heat shock protein 90 (hsp90) and caveolin, substrate and cofactor dependence, and phosphorylation might all affect the level of bioavailable NO. A hypothesis is proposed that the final common pathway of diverse causes of endothelial dysfunction involves abnormalities in eNOS phosphorylation at Ser 1179 and other key phosphorylation sites     

Endothelial nitric oxide synthase gene polymorphism is associated with systemic lupus erythematosus

OBJECTIVE: In systemic lupus erythematosus (SLE), endothelial nitric oxide synthase (eNOS) gene locus has been found to be suggestive of linkage with disease, nitric oxide (NO) is produced in significant amounts, and endothelial cell dysfunction is observed. eNOS gene polymorphism may affect both the synthesis of eNOS protein and its enzymatic activity. We examined the influence of eNOS gene polymorphisms on susceptibility to SLE. METHODS: Genomic DNA from 88 Northwestern Colombian women with SLE, as well as 199 controls matched for sex, age, and ethnicity, was genotyped for the -786T -- > C polymorphism in the promoter region, the intron 4 variable number of tandem repeats, and the Glu298Asp polymorphism in exon 7 of the eNOS gene by polymerase chain reaction and restriction fragment length polymorphism techniques. Haplotype and allele frequency comparisons, a Hardy-Weinberg equilibrium test, and linkage disequilibrium (LD) analysis were performed. RESULTS: The intron 4b allele was associated with SLE (OR 2.2, 95% CI 1.29-3.60, pc = 0.005) as was the 4bb genotype (OR 2.9, 95% CI 1.61-5.33, pc = 0.0009), while the 4a allele was protective (OR 0.4, 95% CI 0.26-0.76, pc = 0.005), as was the 4ab genotype (OR 0.29, 95% CI 0.15-0.56, pc < 0.0001). In controls, all loci were in linkage disequilibrium (p < 0.02). In patients, intron 4 was in Hardy-Weinberg disequilibrium, due to an excess of homozygotes (p = 0.01). CONCLUSION: eNOS polymorphism influences SLE predisposition. Since intron 4bb genotype is responsible for higher levels of eNOS synthesis and intron 4 ab genotype is associated with lower synthesis, our results might provide insight into the elevated levels of NO observed in SLE patients.     

Constitutive nitric oxide synthase activation is a significant route for nitroglycerin-mediated vasodilation

The physiological effects of nitroglycerin as a potent vasodilator have long been documented. However, the molecular mechanisms by which nitroglycerin exerts its biological functions are still a matter of intense debate. Enzymatic pathways converting nitroglycerin to vasoactive compounds have been identified, but none of them seems to fully account for the reported clinical observations. Here, we demonstrate that nitroglycerin triggers constitutive nitric oxide synthase (NOS) activation, which is a major source of NO responsible for low-dose (1-10 nM) nitroglycerin-induced vasorelaxation. Our studies in cell cultures, isolated vessels, and whole animals identified endothelial NOS activation as a fundamental requirement for nitroglycerin action at pharmacologically relevant concentrations in WT animals.     

Inducible nitric oxide synthase and vascular injury

The role nitric oxide (NO) plays in the cardiovascular system is complex and diverse. Even more controversial is the role that the inducible NO synthase enzyme (iNOS) serves in mediating different aspects of cardiovascular pathophysiology. Following arterial injury, NO has been shown to serve many vasoprotective roles, including inhibition of platelet aggregation and adherence to the site of injury, inhibition of leukocyte adherence, inhibition of vascular smooth muscle cell (VSMC) proliferation and migration, and stimulation of endothelial cell (EC) growth. These properties function together to preserve a normal vascular environment following injury. In this review, we discuss what is known about the involvement of iNOS in the vascular injury response. Additionally, we discuss the beneficial role of iNOS gene transfer to the vasculature in preventing the development of neointimal thickening. Lastly, the pathophysiology of transplant vasculopathy is discussed as well as the role of iNOS in this setting.     

Endothelial nitric oxide synthase gene polymorphisms in giant cell arteritis

OBJECTIVE: To examine potential associations of the Glu/Asp(298) polymorphism in exon 7 and the 4a/b polymorphism in intron 4 of the endothelial nitric oxide synthase (eNOS) gene with susceptibility to and clinical expression of giant cell arteritis (GCA), particularly in patients with versus those without ischemic complications. METHODS: Ninety-one consecutive patients with biopsy-proven GCA, who were residents of Reggio Emilia, Italy, and 133 population-based controls from the same geographic area were genotyped by polymerase chain reaction and allele-specific oligonucleotide techniques for eNOS polymorphisms in exon 7 and intron 4. The patients were separated into 2 subgroups according to the presence or absence of ischemic complications (visual loss and/or jaw claudication and/or aortic arch syndrome). RESULTS: The distribution of the Glu/Asp(298) genotype differed significantly between GCA patients and controls (corrected P [P(corr)] = 0.003). Carriers of the Asp(298) allele (Asp/Asp or Glu/Asp) were significantly more frequent among the GCA patients than among the controls (P(corr) = 0.0002, odds ratio 3.3, 95% confidence interval 1.7-6.3). The distribution of the 4a/b genotype was similar in GCA patients and controls. No significant associations were found when GCA patients with and without ischemic complications were compared. CONCLUSION: Our findings show that the Glu/Asp(298) polymorphism of the eNOS gene is associated with GCA susceptibility.