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.

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

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Background Previously, we have shown that endothelial microparticles (EMPs) injected into mice induce acute lung injury (ALI) [1]. In this study, we hypothesize that EMPs induce ALI by initiating cytokine release in the lung, leading to recruitment and activation of neutrophils. Materials and methods C57BL/6J male mice (8–10 wk old) were intravenously injected with EMPs (200,000/mL), LPS (2 mg/kg), or both. Bronchoalveolar lavage (BAL) and serum levels of IL-1β and TNF-α were analyzed by enzyme-linked immunoassay (ELISA). Morphometric analysis was performed on H and E stained lung sections. Myeloperoxidase (MPO) levels were determined via an enzymatic assay and immunofluorescence of stained sections. Results EMPs led to significantly increased pulmonary and systemic IL-1β and TNF-α levels, which correlated with increased neutrophil recruitment to the lung. MPO levels in the lungs were increased significantly following injection of EMPs or LPS, compared to PBS. In mice treated with EMPs and LPS either simultaneously or successively, the cytokine and MPO levels were significantly increased over that of either treatment alone. Conclusion EMPs contribute to lung injury through the initiation of a cytokine cascade that increases recruitment of neutrophils and subsequent release of MPO. Furthermore, treatment of mice with both EMPs and LPS induced greater lung injury than either treatment alone, suggesting that EMPs prime the lung for increased injury by other pathogens. Therapies aimed at reducing or blocking EMPs may be a useful strategy for attenuating lung injury.