Jingsong Ou

L-4F, an Apolipoprotein A-1 Mimetic, Dramatically Improves Vasodilation in Hypercholesterolemia and Sickle Cell Disease

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 (O2·−) generation. Recent reports indicate that apolipoprotein (apo) A-1 mimetics inhibit atherosclerosis in LDL receptor–null (Ldlr−/−) 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 O2·− generation. Methods and Results—Arterioles were isolated from hypercholesterolemic Ldlr−/− 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 O2·− 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−/− mice, which L-4F dramatically improved. L-4F inhibited LDL-induced increases in O2·− 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.

Comparative proteomic analysis of PAI-1 and TNF-alpha-derived endothelial microparticles

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.

L-4F, an Apolipoprotein A-1 Mimetic, Restores Nitric Oxide and Superoxide Anion Balance in Low-Density Lipoprotein-Treated Endothelial Cells

Background—Low-density lipoprotein (LDL) impairs endothelial cell function by uncoupling endothelial nitric oxide synthase (eNOS) activity, which allows superoxide anion (O2·−) 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 LDL±L-4F, and changes in A23187-stimulated ·NO and O2·− generation were determined by ozone chemiluminescence and superoxide dismutase-inhibitable 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) interactions with eNOS. LDL decreased ·NO production and increased eNOS-dependent O2·− generation. Pretreatment of LDL with L-4F increased ·NO and decreased O2·− generation. By itself, L-4F had no effect on O2·− but did increase ·NO generation. Stimulation of endothelial cells incubated with LDL decreased the association of hsp90 with eNOS. Pretreatment of LDL with L-4F prevented a decrease in hsp90 association with eNOS and often enhanced association on stimulation. Conclusions—These data demonstrate that L-4F protects endothelial cell function by preventing LDL from uncoupling eNOS activity. L-4F allows endothelial cell to maintain coupled eNOS activity to generate ·NO even in the face of atherogenic concentrations of LDL.

ENDOTHELIUM-DERIVED MICROPARTICLES INDUCE ENDOTHELIAL DYSFUNCTION AND ACUTE LUNG INJURY

ABSTRACT 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 × 106/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.

Inhibition of heat shock protein 90 (hsp90) in proliferating endothelial cells uncouples endothelial nitric oxide synthase activity.

Dual increases in nitric oxide ((*)NO) and superoxide anion (O(2)(*-)) production are one of the hallmarks of endothelial cell proliferation. Increased expression of endothelial nitric oxide synthase (eNOS) has been shown to play an important role in maintaining high levels of (*)NO generation to offset the increase in O(2)(*-) that occurs during proliferation. Although recent reports indicate that heat shock protein 90 (hsp90) associates with eNOS to increase (*)NO generation, the role of hsp90 association with eNOS during endothelial cell proliferation remains unknown. In this report, we examine the effects of endothelial cell proliferation on eNOS expression, hsp90 association with eNOS, and the mechanisms governing eNOS generation of (*)NO and O(2)(*-). Western analysis revealed that endothelial cells not only increased eNOS expression during proliferation but also hsp90 interactions with the enzyme. Pretreatment of cultures with radicicol (RAD, 20 microM), a specific inhibitor that does not redox cycle, decreased A23187-stimulated (*)NO production and increased L(omega)-nitroargininemethylester (L-NAME)-inhibitable O(2)(*-) generation. In contrast, A23187 stimulation of controls in the presence of L-NAME increased O(2)(*-) generation, confirming that during proliferation eNOS generates (*)NO. Our findings demonstrate that hsp90 plays an important role in maintaining (*)NO generation during proliferation. Inhibition of hsp90 in vascular endothelium provides a convenient mechanism for uncoupling eNOS activity to inhibit (*)NO production. This study provides new understanding of the mechanisms by which ansamycin antibiotics inhibit endothelial cell proliferation. Such information may be useful in the development and design of new antineoplastic agents in the future.

Angiostatin A Negative Regulator of Endothelial-Dependent Vasodilation

Background—Angiostatin is known to inhibit certain aspects of endothelial function, eg, angiogenesis. Here we investigated the effects of angiostatin on another aspect of endothelial function, vasodilation, and examined mechanisms of inhibition—namely, association of heat-shock protein 90 (hsp90) with endothelial nitric oxide synthase (eNOS) and endothelial generation of nitric oxide (·NO) and superoxide anion (&OV0151;). This avenue of investigation was based on recent reports suggesting that hsp90 modulates NOS production of ·NO and &OV0151;. Methods and Results—Effects of angiostatin on vasodilation were determined in arterioles with the use of videomicroscopy in response to endothelium- and ·NO-dependent vasodilators, acetylcholine (ACh) and vascular endothelial growth factor (VEGF), and an endothelium-independent agonist, papaverine. Association of hsp90 with eNOS was determined in rat aortas and bovine aortic endothelial cells (BAECs). Effects of angiostatin on ·NO and &OV0151; generation by BAECs were determined by ozone chemiluminescence and superoxide dismutase (SOD)–inhibitable ferricytochrome c reduction, respectively. Angiostatin impaired vasodilation mediated by ACh and VEGF but not papaverine. Pretreating arterioles with polyethylene glycolated–SOD (PEG-SOD) improved vasodilation to ACh and VEGF. Angiostatin decreased the association of hsp90 with eNOS in aortas and BAEC cultures and increased &OV0151; generation in stimulated BAECs by an L&ggr;-nitroargininemethylester (L-NAME)–inhibitable mechanism. Conclusions—These data indicate angiostatin alters endothelial function by allowing eNOS to generate &OV0151; on activation. Such changes in enzyme function begin to explain, in part, why angiostatin is antiangiogenic and impairs endothelium-dependent vasodilation.

Native low-density lipoprotein induces endothelial nitric oxide synthase dysfunction: role of heat shock protein 90 and caveolin-1.

Although native LDL (n-LDL) is well recognized for inducing endothelial cell (EC) dysfunction, the mechanisms remain unclear. One hypothesis is n-LDL increases caveolin-1 (Cav-1), which decreases nitric oxide (*NO) production by binding endothelial nitric oxide synthase (eNOS) in an inactive state. Another is n-LDL increases superoxide anion (O(2)(*-)), which inactivates *NO. To test these hypotheses, EC were incubated with n-LDL and then analyzed for *NO, O(2)(*-), phospho-eNOS (S1179), eNOS, Cav-1, calmodulin (CaM), and heat shock protein 90 (hsp90). n-LDL increased NOx by more than 4-fold while having little effect on A23187-stimulated nitrite production. In contrast, n-LDL decreased cGMP under basal and A23187-stimulated conditions and increased O(2)(*-) by a mechanism that could be inhibited by L-nitroargininemethylester (L-NAME) and BAPTA/AM. n-LDL increased phospho-eNOS by 149%, eNOS by approximately 34%, and Cav-1 by 28%, and decreased the association of hsp90 with eNOS by 49%. n-LDL did not appear to alter eNOS distribution between membrane fractions (approximately 85%) and cytosol (approximately 15%). Only 3-6% of eNOS in membrane fractions was associated with Cav-1. These data support the hypothesis that n-LDL increases O(2)(*-), which scavenges *NO, and suggest that n-LDL uncouples eNOS activity by decreasing the association of hsp90 as an initial step in signaling eNOS to generate O(2)(*-).

AP-4F, antennapedia peptide linked to an amphipathic α helical peptide, increases the efficiency of Lipofectamine-mediated gene transfection in endothelial cells

Typically, endothelial cells are difficult to transfect. In this study, we report that antennapedia peptide (AP) linked to L-4F, a water-soluble, amphipathic alpha helical peptide that avidly binds lipids (AP-4F) increases Lipofectamine 2000-mediated transfection of bovine coronary endothelial cell cultures. Transfection efficiency was monitored by flow cytometry and fluorescent microscopy. Lipofectamine 2000 transfection of endothelial cell cultures with green fluorescence protein (GFP)-DNA typically yields transfection efficiencies of 35.4+/-3.3% with low levels of cell death (8.1+/-1.0%). Pre-treatment of the Lipofectamine 2000-GFP-DNA complexes with AP-4F for 5 min increased transfection to 58.2+/-2.8% without increasing cell death. AP-4F increases Lipofectamine 2000-mediated transfection in a time-dependent fashion (within 10-20 min). Systematic studies reveal that the individual components of AP-4F, i.e., AP and L-4F alone, are ineffective in increasing Lipofectamine 2000-mediated transfection and that AP-4F must be directly associated with DNA liposomes prior to transfection for optimal uptake by endothelial cells. These observations demonstrate that AP-4F may be useful for increasing the transfection efficiency of endothelial cell cultures with standard commercially available reagents.