. Decreased association of HSP90 impairs endothelial nitric oxide synthase in fetal lambs with persistent pulmonary hypertension. Am J Physiol Heart Circ Physiol 285: H204-H211, 2003; 10.1152/ajpheart.00837. 2002.-Persistent pulmonary hypertension of newborn (PPHN) is associated with decreased nitric oxide (NO) release and impaired pulmonary vasodilation. We investigated the hypothesis that decreased association of heat shock protein 90 (HSP90) with endothelial NO synthase (eNOS) impairs NO release and vasodilation in PPHN. The responses to the NOS agonist ATP were investigated in fetal lambs with PPHN induced by prenatal ligation of ductus arteriosus, and in sham ligation controls. ATP caused dose-dependent vasodilation in control pulmonary resistance arteries, and this response was attenuated in PPHN vessels. The response of control pulmonary arteries to ATP was attenuated by N Gnitro-L-arginine methyl ester (L-NAME), a NOS antagonist, and geldanamycin, an inhibitor of HSP90-eNOS interaction. The attenuated response to ATP observed in PPHN was improved by pretreatment of vessels with L-NAME or 4,5-dihydroxy-1,3-benzene-disulfonate, a superoxide scavenger. Pulmonary arteries from PPHN lambs had decreased basal levels of HSP90 in association with eNOS. Association of HSP90 with eNOS and NO release increased in response to ATP in control pulmonary artery endothelial cells, but not in cells from PPHN lambs. Decreased HSP90-eNOS interactions may contribute to the impaired NO release and vasodilation observed in the ductal ligation model of PPHN. persistent pulmonary hypertension; ATP; newborn A RAPID AND SUSTAINED DECREASE in the pulmonary vascular resistance at birth facilitates initiation of gas exchange during postnatal life. Release of endothelium-derived nitric oxide (NO) in the pulmonary circulation plays a major role in this birth-related vasodilation (2, 34, 40). Failure of this adaptation results in persistent pulmonary hypertension of newborn (PPHN), a condition associated with decreased NO synthase (NOS) activity (5, 35, 41), expression (5, 35, 41), and impaired 38) in fetal pulmonary arteries. The mechanism(s) of impaired NOS activity and pulmonary vasodilation in PPHN remains unknown. Our laboratory's previous studies (15-18) demonstrated that the purine nucleotide ATP contributes to birth-related pulmonary vasodilation in fetal lambs. ATP causes pulmonary vasodilation in fetal lambs in part by stimulation of NO release (17). ATP stimulates NO release from cultured vascular endothelial cells (8,28). However, the mechanism of NOS activation by ATP and its alteration in PPHN remain unknown.Heat shock protein 90 (HSP90), a molecular chaperone, modulates the endothelial NOS (eNOS) activity (10, 12, 32) and balance of NO and superoxide (O 2 Ϫ ⅐) release from eNOS (30) in response to several physiological stimuli. However, the role of this constitutively expressed chaperone in mediating vasodilation in the fetal pulmonary arteries and its role in mediating the effects of ATP on eNOS is unknown. We propose...

Acute lung injury (ALI) carries a high mortality in critically ill patients. Recent reports correlate elevated concentrations of endothelium-derived microparticles (EMPs) with diseases of endothelial dysfunction. Many of these diseases have ALI sequelae. We hypothesize that EMPs contribute to endothelial cell (EC) dysfunction and development of ALI. To test this hypothesis, we treated isolated vessels with EMPs and examined changes in vasodilation. Endothelial cell cultures were incubated with EMPs and examined for changes in stimulated nitric oxide (*NO) production and nitric oxide synthase (eNOS) activation. Finally, EMPs were injected into rats and mice and lungs examined for ALI. In both mouse and human ex vivo vessel preparations, we found a marked attenuation of endothelium-mediated vasodilation after EMP treatment (4 x 10(6)/mL). This dysfunction was not corrected by pretreatment of EMPs with free radical scavengers. Coincubation of EMPs with EC cultures yielded a three-fold reduction in A23187-stimulated *NO release. Western analysis of these cells showed a corresponding decrease in eNOS phosphorylation at Ser1179 and a decrease in hsp90 association. Measurements of lung permeability, myeloperoxidase activity, and histology of EMPs-treated Brown Norway rats demonstrated pulmonary edema, neutrophil recruitment, and compromise of the endothelial-alveolar barrier as a second hit phenomenon. In C57BL/6 mice, exogenous EMPs caused a significant rise in pulmonary capillary permeability both as a primary and secondary injury. These findings demonstrate EMPs are capable of inducing significant lung injury at pathophysiologically relevant concentrations. Endothelium-derived microparticles inhibit endothelium-mediated vasodilation and *NO generation from eNOS. Once elucidated, EMP mechanisms of inducing ALI and endothelial dysfunction may present new therapeutic targets.

Nitric oxide (NO), generated from L-arginine by endothelial nitric oxide synthase (eNOS), is a key endothelial-derived factor whose bioavailability is essential to the normal function of the endothelium. Endothelium dysfunction is characterized by loss of NO bioavailability because of either reduced formation or accelerated degradation of NO. We have recently reported that overexpression of vascular cytochrome P-450 (CYP) 4A in rats caused hypertension and endothelial dysfunction driven by increased production of 20-hydroxyeicosatetraenoic acid (20-HETE), a major vasoconstrictor eicosanoid in the microcirculation. To further explore cellular mechanisms underlying CYP4A-20-HETE-driven endothelial dysfunction, the interactions between 20-HETE and the eNOS-NO system were examined in vitro. Addition of 20-HETE to endothelial cells at concentrations as low as 1 nM reduced calcium ionophore-stimulated NO release by 50%. This reduction was associated with a significant increase in superoxide production. The increase in superoxide in response to 20-HETE was prevented by N(G)-nitro-L-arginine methyl ester, suggesting that uncoupled eNOS is a source of this superoxide. The response to 20-HETE was specific in that 19-HETE did not affect NO or superoxide production, and, in fact, the response to 20-HETE could be competitively antagonized by 19(R)-HETE. 20-HETE had no effect on phosphorylation of eNOS protein at serine-1179 or threonine-497 following addition of calcium ionophore; however, 20-HETE inhibited association of eNOS with 90-kDa heat shock protein (HSP90). In vivo, impaired acetylcholine-induced relaxation in arteries overexpressing CYP4A was associated with a marked reduction in the levels of phosphorylated vasodilator-stimulated phosphoprotein, an indicator of bioactive NO, that was reversed by inhibition of 20-HETE synthesis or action. Because association of HSP90 with eNOS is critical for eNOS activation and coupled enzyme activity, inhibition of this association by 20-HETE may underlie the mechanism, at least in part, by which increased CYP4A expression and activity cause endothelial dysfunction.

Objective-The goal of this study was to determine the expression signature and the potential role of microRNAs in human arteries with arteriosclerosis obliterans (ASO). Methods and Results-The expression profiles of microRNAs in human arteries with ASO and in normal control arteries were determined by quantitative reverse transcription-polymerase chain reaction array. Among the 617 detected microRNAs, multiple microRNAs were aberrantly expressed in arteries with ASO. Some of these dysregulated microRNAs were further verified by quantitative reverse transcription-polymerase chain reaction. Among them, microRNA-21 (miR-21) was mainly located in arterial smooth muscle cells (ASMCs) and was increased by more than 7-fold in ASO that was related to hypoxia inducible factor 1-␣. In cultured human ASMCs, cell proliferation and migration were significantly decreased by inhibition of miR-21. 3Ј-Untranslated region luciferase assay confirmed that tropomyosin 1 was a target of miR-21 that was involved in miR-21-mediated cellular effects, such as cell shape modulation. Key Words: atherosclerosis Ⅲ hypoxia Ⅲ peripheral arterial disease Ⅲ vascular muscle Ⅲ microRNA A rteriosclerosis obliterans (ASO) of the lower extremities is a major cause of adult limb loss worldwide. 1-3 Surgery is still the major approach in the treatment of ASO. However, many patients develop restenosis in 1 year after surgery. 1 It is well established that proliferation and migration of arterial smooth muscle cells (ASMCs) are the major cellular events and the major reasons behind ASO formation and posttreatment restenosis. 4 However, the molecular mechanisms involved in regulation of proliferation and migration of ASMCs in ASO remain unclear. Conclusion-The See accompanying articles on pages 1939 and 1941MicroRNAs are a novel class of endogenous, small noncoding RNAs that regulate approximately 30% of the encoding genes of the human genome at the posttranscriptional level by binding the 3Ј-untranslated region (UTR) of their target mRNAs. [5][6][7] The microRNA expression profile in vessels has recently been described by Ji et al 8 Several microRNAs, including microRNA-21 (miR-21), miR-221/222, and miR-145, have been found to modulate ASMC function and be involved in the process of artery stenosis in the rat carotid artery balloon injury model. 8 -10 However, the expression profiles of microRNAs in human arteries with ASO are still unknown.Tissue-specific expression is an important characteristic of microRNA expression. 11 For example, miR-1 is highly expressed in heart, but its expression in artery is low. 8 Such different expression profiles in different tissues suggest that the physiological functions of microRNA in different tissues may be different. 8 Although human ASO and artery stenosis in rat carotid artery injury models share many features, their pathological processes are different. 12,13 Thus, identifying microRNAs in ASO and clarifying their biological functions would be useful for understanding the mechanisms of ASO formation and searc...

Abstract-Previously we showed L-4F, a novel apolipoprotein A-I (apoA-I) mimetic, improved vasodilation in 2 dissimilar models of vascular disease: hypercholesterolemic LDL receptor-null (Ldlr Ϫ/Ϫ ) mice and transgenic sickle cell disease mice. Here we determine the mechanisms by which D-4F improves vasodilation and arterial wall thickness in hypercholesterolemic Ldlr Ϫ/Ϫ mice and Ldlr Ϫ/Ϫ /apoA-I null (apoA-I Ϫ/Ϫ ), double-knockout mice. Ldlr Ϫ/Ϫ and Ldlr Ϫ/Ϫ / apoA-I Ϫ/Ϫ mice were fed Western diet (WD) with and without D-4F. Oral D-4F restored endothelium-and endothelial NO synthase (eNOS)-dependent vasodilation in direct relationship to duration of treatments and reduced wall thickness in as little as 2 weeks in vessels with preexisting disease in Ldlr Ϫ/Ϫ mice. D-4F had no effect on total or HDL cholesterol concentrations but reduced proinflammatory HDL levels. D-4F had no effect on plasma myeloperoxidase concentrations but reduced myeloperoxidase association with apoA-I as well as 3-nitrotyrosine in apoA-I. D-4F increased endothelium-and eNOS-dependent vasodilation in Ldlr Key Words: cardiovascular diseases Ⅲ hypercholesterolemia Ⅲ lipoproteins Ⅲ nitric oxide synthase Ⅲ vasodilation E ndothelial cells play an important role in maintaining vascular health. One of the earliest physiological and most sensitive changes in hypercholesterolemia is loss of endothelium-and endothelial NO synthase (eNOS)-dependent vasodilation, which appears to occur before structural changes in the vessel wall. 1,2 In a prospective human study, angiography was used to observe impaired endothelium-dependent vasodilation in coronary arteries and was found to be a strong prognostic indicator of vascular pathology. 3 Such findings, 3 as well as those from others, 4,5 support the concept that endothelium-dependent vasodilation provides important insights into the atherogenic state of the vessel wall.We previously reported L-4F, when administered by IP injection, increased eNOS-dependent vasodilation of small arteries from hypercholesterolemic Ldlr Ϫ/Ϫ mice and from transgenic sickle cell mice. 6 D-4F is the same as L-4F, except it is synthesized from D-amino acids. In the D conformation, 4F is resistant to metabolism compared with 4F synthesized from L-amino acids, which is rapidly degraded after oral administration. 8 Oral D-4F reduced lesions in Ldlr Ϫ/Ϫ mice fed WD by more than 79% by restoring HDL function without significantly altering cholesterol or HDL cholesterol levels. 7 Interestingly, D-4F also reduced lesions in apoE Ϫ/Ϫ mice, which were hypercholesterolemic from birth. 7 However, to date, no studies have been performed to determine Original

Endothelium-derived microparticles (EMPs) are small vesicles released from endothelial cells in response to cell injury, apoptosis, or activation. Elevated concentrations of EMPs have been associated with many inflammatory and vascular diseases. EMPs also mediate long range signaling and alter downstream cell function. Unfortunately, the molecular and cellular basis of microparticle production and downstream cell function is poorly understood. We hypothesize that EMPs generated by different agonists will produce distinct populations of EMPs with unique protein compositions. To test this hypothesis, different EMP populations were generated from human umbilical vein endothelial cells by stimulation with plasminogen activator inhibitor type 1 (PAI-1) or tumor necrosis factor-alpha (TNF-α) and subjected to proteomic analysis by LC/MS. We identified 432 common proteins in all EMP populations studied. Also identified were 231 proteins unique to control EMPs, 104 proteins unique to PAI-1 EMPs and 70 proteins unique to TNF-α EMPs. Interestingly, variations in protein abundance were found among many of the common EMP proteins, suggesting that differences exist between EMPs on a relative scale. Finally, gene ontology (GO) and KEGG pathway analysis revealed many functional similarities and few differences between the EMP populations studied. In summary, our results clearly indicate that EMPs generated by PAI-1 and TNF-α produce EMPs with overlapping but distinct protein compositions. These observations provide fundamental insight into the mechanisms regulating the production of these particles and their physiological role in numerous diseases.

Background-Hypercholesterolemia and sickle cell disease (SCD) impair endothelium-dependent vasodilation by dissimilar mechanisms. Hypercholesterolemia impairs vasodilation by a low-density lipoprotein (LDL)-dependent mechanism. SCD has been characterized as a chronic state of inflammation in which xanthine oxidase (XO) from ischemic tissues increases vascular superoxide anion (O 2 ·Ϫ ) generation. Recent reports indicate that apolipoprotein (apo) A-1 mimetics inhibit atherosclerosis in LDL receptor-null (Ldlr Ϫ/Ϫ ) mice fed Western diets. Here we hypothesize that L-4F, an apoA-1 mimetic, preserves vasodilation in hypercholesterolemia and SCD by decreasing mechanisms that increase O 2 ·Ϫ generation. Methods and Results-Arterioles were isolated from hypercholesterolemic LdlrϪ/Ϫ mice and from SCD mice that were treated with either saline or L-4F (1 mg/kg per day). Vasodilation in response to acetylcholine was determined by videomicroscopy. Effects of L-4F on LDL-induced increases in endothelium-dependent O 2 ·Ϫ generation were determined on arterial segments via the hydroethidine assay and on stimulated endothelial cell cultures via superoxide dismutase-inhibitable ferricytochrome c reduction. Effects of L-4F on XO bound to pulmonary arterioles and content in livers of SCD mice were determined by immunofluorescence. Hypercholesterolemia impaired vasodilation in Ldlr Ϫ/Ϫ mice, which L-4F dramatically improved. L-4F inhibited LDL-induced increases in O 2 ·Ϫ in arterial segments and in stimulated cultures. SCD impaired vasodilation, increased XO bound to pulmonary endothelium, and decreased liver XO content. L-4F dramatically improved vasodilation, decreased XO bound to pulmonary endothelium, and increased liver XO content compared with levels in untreated SCD mice. Conclusions-These data show that L-4F protects endothelium-dependent vasodilation in hypercholesterolemia and SCD.Our findings suggest that L-4F restores vascular endothelial function in diverse models of disease and may be applicable to treating a variety of vascular diseases. Clinical studies clearly indicate that HDL plays an important role in protecting vascular function against atherosclerosis. 8 Transgenic expression of apoA-1, the major atheroprotective apolipoprotein of HDL, retards the progression of advanced lesions in transplanted aortas from apoE-null mice and remodels them to a more stable-appearing phenotype. 9 Intraperitoneal injection of an apoA-1 mimetic (5F) and parental administration of another apoA-1 mimetic (D-4F) enhances the ability of HDL to inhibit LDL oxidation and to protect mice from diet-induced atherosclerosis without changing plasma cholesterol levels. 6,10 Indeed, infusion of reconstituted HDL rapidly improves endothelium-and endothelial nitric oxide synthase (eNOS)-dependent forearm blood flow in hypercholesterolemic men, 11 confirming that HDL plays a critical role in protecting endothelial cell function.The mechanisms by which SCD have been shown to impair vasodilation at first glance appear distinctly different from tho...

Background-Low-density lipoprotein (LDL) impairs endothelial cell function by uncoupling endothelial nitric oxide synthase (eNOS) activity, which allows superoxide anion (O 2 ·Ϫ ) to be generated rather than nitric oxide (·NO). Recent reports indicate that apolipoprotein (apo) A-1 mimetics inhibit the development of atherosclerotic lesions in LDL receptor-null mice. Here we hypothesize that L-4F, an apoA-1 mimetic that inhibits atherosclerosis induced by hypercholesterolemia, protects endothelial cell function by preventing LDL from uncoupling eNOS activity. Methods and Results-Bovine aortic endothelial cells were incubated with LDLϮL-4F, and changes in A23187-stimulated ·NO and O 2 ·Ϫ generation were determined by ozone chemiluminescence and superoxide dismutaseinhibitable ferricytochrome c reduction, respectively. Western analysis of eNOS immunoprecipitates was used to determine effects of LDL and L-4F on heat shock protein 90 (hsp90)