Pcpe2, a Novel Extracellular Matrix Protein, Regulates Adipocyte SR-BI-Mediated High-Density Lipoprotein Uptake.

Objective: To investigate the role of adipocyte Pcpe2 (procollagen C-endopeptidase enhancer 2) in SR-BI (scavenger receptor class BI)-mediated HDL-C (high-density lipoprotein cholesterol) uptake and contributions to adipose lipid storage. Approach and Results: Pcpe2, a glycoprotein devoid of intrinsic proteolytic activity, is believed to participate in extracellular protein-protein interactions, supporting SR-BI- mediated HDL-C uptake. In published studies, Pcpe2 deficiency increased the development of atherosclerosis by reducing SR-BI-mediated HDL-C catabolism, but the biological impact of this deficiency on adipocyte SR-BI-mediated HDL-C uptake is unknown. Differentiated cells from Ldlr-/-/Pcpe2-/- (Pcpe2-/-) mouse adipose tissue showed elevated SR-BI protein levels, but significantly reduced HDL-C uptake compared to Ldlr-/- (control) adipose tissue. SR-BI-mediated HDL-C uptake was restored by preincubation of cells with exogenous Pcpe2. In diet-fed mice lacking Pcpe2, significant reductions in visceral, subcutaneous, and brown adipose tissue mass were observed, despite elevations in plasma triglyceride and cholesterol concentrations. Significant positive correlations exist between adipose mass and Pcpe2 expression in both mice and humans. Conclusions: Overall, these findings reveal a novel and unexpected function for Pcpe2 in modulating SR-BI expression and function as it relates to adipose tissue expansion and cholesterol balance in both mice and humans.

Sickle cell disease increases high mobility group box 1: a novel mechanism of inflammation.

High mobility group box 1 (HMGB1) is a chromatin-binding protein that maintains DNA structure. On cellular activation or injury, HMGB1 is released from activated immune cells or necrotic tissues and acts as a damage-associated molecular pattern to activate Toll-like receptor 4 (TLR4). Little is known concerning HMGB1 release and TLR4 activity and their role in the pathology of inflammation of sickle cell disease (SCD). Circulating HMGB1 levels were increased in both humans and mice with SCD compared with controls. Furthermore, sickle plasma increased HMGB1-dependent TLR4 activity compared with control plasma. HMGB1 levels were further increased during acute sickling events (vasoocclusive crises in humans or hypoxia/reoxygenation injury in mice). Anti-HMGB1 neutralizing antibodies reduced the majority of sickle plasma-induced TLR4 activity both in vitro and in vivo. These findings show that HMGB1 is the major TLR4 ligand in SCD and likely plays a critical role in SCD-mediated inflammation.

Role of autophagy in angiogenesis in aortic endothelial cells.

Angiogenesis plays critical roles in the recovery phase of ischemic heart disease and peripheral vascular disease. An increase in autophagy is protective under hypoxic and chronic ischemic conditions. In the present study we determined the role of autophagy in angiogenesis. 3-Methyladenine (3-MA) and small interfering RNA (siRNA) against ATG5 were used to inhibit autophagy induced by nutrient deprivation of cultured bovine aortic endothelial cells (BAECs). Assays of BAECs tube formation and cell migration revealed that inhibition of autophagy by 3-MA or siRNA against ATG5 reduced angiogenesis. In contrast, induction of autophagy by overexpression of ATG5 increased BAECs tube formation and migration. Additionally, inhibiting autophagy impaired vascular endothelial growth factor (VEGF)-induced angiogenesis. However, inhibition of autophagy did not alter the expression of pro-angiogenesis factors such as VEGF, platelet-derived growth factor, or integrin αV. Furthermore, autophagy increased reactive oxygen species (ROS) formation and activated AKT phosphorylation. Inhibition of autophagy significantly decreased the production of ROS and activation of AKT but not of extracellular regulated kinase, whereas overexpression of ATG5 increased cellular ROS production and AKT activation in BAECs. Inhibition of AKT activation or ROS production significantly decreased the tube formation induced by ATG5 overexpression. Here we report a novel observation that autophagy plays an important role in angiogenesis in BAECs. Induction of autophagy promotes angiogenesis while inhibition of autophagy suppresses angiogenesis, including VEGF-induced angiogenesis. ROS production and AKT activation might be important mechanisms for mediating angiogenesis induced by autophagy. Our findings indicate that targeting autophagy may provide an important new tool for treating cardiovascular disease.

Decreases in manganese superoxide dismutase expression and activity contribute to oxidative stress in persistent pulmonary hypertension of the newborn.

A rapid increase in the synthesis and release of nitric oxide (NO) facilitates the pulmonary vasodilation that occurs during birth-related transition. Alteration of this transition in persistent pulmonary hypertension of the newborn (PPHN) is associated with impaired function of endothelial nitric oxide synthase (eNOS) and an increase in oxidative stress. We investigated the hypothesis that a decrease in expression and activity of mitochondrial localized manganese superoxide dismutase (MnSOD) in pulmonary artery endothelial cells (PAEC) increases oxidative stress and impairs eNOS function in PPHN. We isolated PAEC and pulmonary arteries from fetal lambs with PPHN induced by prenatal ductus arteriosus ligation or sham ligation (control). We investigated MnSOD expression and activity, tyrosine nitration of MnSOD, and mitochondrial O(2)(-) levels in PAEC from control and PPHN lambs. We introduced exogenous MnSOD via an adenoviral vector (ad-MnSOD) transduction into PAEC and pulmonary arteries of PPHN lambs. The effect of ad-MnSOD was investigated on: mitochondrial O(2)(-) levels, MnSOD and eNOS expression and activity, intracellular hydrogen peroxide (H(2)O(2)) levels, and catalase expression in PAEC. MnSOD mRNA and protein levels and activity were decreased and MnSOD tyrosine nitration was increased in PPHN-PAEC. ad-MnSOD transduction of PPHN-PAEC increased its activity two- to threefold, decreased mitochondrial O(2)(-) levels, and increased H(2)O(2) levels and catalase expression. ad-MnSOD transduction improved eNOS expression and function and the relaxation response of PPHN pulmonary arteries. Our observations suggest that decreased MnSOD expression and activity contribute to the endothelial dysfunction observed in PPHN.

Endothelial microparticles induce inflammation in acute lung injury.

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-1ß 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-1ß 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.

Mesenteric nitric oxide and superoxide production in experimental necrotizing enterocolitis.

BACKGROUND: A proposed mechanism of intestinal injury in necrotizing enterocolitis (NEC) involves vascular dysfunction through altered nitric oxide synthase (NOS) activity. We hypothesize that this dysfunction results in an imbalance in nitric oxide (*NO) and superoxide (O(2)(*-)) production by the intestinal vascular endothelium, which contributes to the intestinal injury seen in NEC. MATERIALS AND METHODS: Neonatal rat pups were divided into two groups. Control pups were breast fed and housed with their mother. Experimental NEC pups were housed separately and either exposed to formula feeding and 5% to 10% hypoxia alone (FF/H) or with the addition of lipopolysaccharide (FF/H/LPS). Mesenteries from each group were analyzed for *NO and O(2)(*-) production with and without NOS inhibition by N(G)-monomethyl-L-arginine (L-NMMA). Western blot analysis for eNOS, phosphorylated eNOS (phospho-eNOS), and inducible NOS (iNOS) was performed, and each terminal ileum was graded for intestinal injury by histology. RESULTS: Histology revealed mild intestinal injury (grade 1-2 on a 4-point scale) in the FF/H group and severe injury (grade 3-4) in the FF/H/LPS group. The FF/H cohort had significantly increased *NO and lower O(2)(*-) production, while the FF/H/LPS group shifted to significantly decreased *NO and increased O(2)(*-) production. L-NMMA inhibited >50% of O(2)(*-) production in all three groups but only inhibited *NO production in control and FF/H pups. Western blot analysis revealed increased levels of phospho-eNOS in FF/H pups and increased iNOS in FF/H/LPS pups. CONCLUSIONS: This study demonstrates in the progression of NEC, intestinal ischemia is associated with a shift from *NO to O(2)(*-) production, which is NOS-dependent. Potentially greater injury results from impaired vasodilatation and over-production of reactive oxygen species.

Integrating Mouse and Human Genetic Data to Move beyond GWAS and Identify Causal Genes in Cholesterol Metabolism.

Identifying the causal gene(s) that connects genetic variation to a phenotype is a challenging problem in genome-wide association studies (GWASs). Here, we develop a systematic approach that integrates mouse liver co-expression networks with human lipid GWAS data to identify regulators of cholesterol and lipid metabolism. Through our approach, we identified 48 genes showing replication in mice and associated with plasma lipid traits in humans and six genes on the X chromosome. Among these 54 genes, 25 have no previously identified role in lipid metabolism. Based on functional studies and integration with additional human lipid GWAS datasets, we pinpoint Sestrin1 as a causal gene associated with plasma cholesterol levels in humans. Our validation studies demonstrate that Sestrin1 influences plasma cholesterol in multiple mouse models and regulates cholesterol biosynthesis. Our results highlight the power of combining mouse and human datasets for prioritization of human lipid GWAS loci and discovery of lipid genes.

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-alpha) 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-alpha 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-alpha 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.

Protein composition of plasminogen activator inhibitor type 1-derived endothelial microparticles.

Endothelial microparticles (EMPs) are small vesicles released from the plasma membrane of endothelial cells in response to cell injury, apoptosis, or activation. Low levels of MPs are shed into the blood from the endothelium, but in some pathologic states, the number of EMPs is elevated. The mechanism of MP formation and the wide-ranging effects of elevated EMPs are poorly understood. Here, we report the protein composition of EMPs derived from human umbilical cord endothelial cells stimulated with plasminogen activator inhibitor type 1 (PAI-1). Two-dimensional gel electrophoresis followed by mass spectrometry identified 58 proteins, of which some were verified by Western blot analysis. Gene Ontology database searches revealed that proteins identified on PAI-1-derived EMPs are highly diverse. Endothelial microparticles are composed of proteins from different cellular components that exhibit multiple molecular functions and are involved in a variety of biological processes. Important insight is provided into the generation and protein composition of PAI-1-derived EMPs.

Effects of D-4F on vasodilation and vessel wall thickness in hypercholesterolemic LDL receptor-null and LDL receptor/apolipoprotein A-I double-knockout mice on Western diet.

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(-/-)) 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(-/-) mice and Ldlr(-/-)/apoA-I null (apoA-I(-/-)), double-knockout mice. Ldlr(-/-) and Ldlr(-/-)/apoA-I(-/-) 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(-/-) 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(-/-)/apoA-I(-/-) mice but did not reduce wall thickness as it had in Ldlr(-/-) mice. Vascular endothelial cells were treated with 22(R)-hydroxycholesterol with and without L-4F. 22(R)-Hydroxycholesterol decreased NO (*NO) and increased superoxide anion (O2*-) production and increased ATP-binding cassette transporter-1 and collagen expression. L-4F restored *NO and O2*- balance, had little effect on ATP-binding cassette transporter-1 expression, but reduced collagen expression. These data demonstrate that although D-4F restores vascular endothelial cell and eNOS function to increase vasodilation, HDL containing apoA-I, or at least some critical concentration of the antiatherogenic lipoprotein, is required for D-4F to decrease vessel wall thickness.

Endothelium-derived microparticles inhibit human cardiac valve endothelial cell function.

Elevated numbers of endothelium-derived microparticles (EMPs) in the circulation are found in a variety of critical illnesses. EMPs have been associated with vascular dysfunction, including thrombotic complications and loss of normal vascular reactivity, common responses associated with cardiac valve injury. However, the exact mechanisms of this dysfunction and the potential impact on cardiac endothelium are unknown. We hypothesize that pathologic levels of circulating EMPs negatively regulate proliferation and migration of valvular endothelial cells (ECs), leading to downstream endothelial dysfunction. EMPs were generated from plasminogen activation inhibitor 1-stimulated human umbilical vein endothelial cells (HUVECs). Human mitral valve endothelial cells (HMVECs) were isolated and characterized by platelet endothelial cell-derived adhesion molecule-1 (PECAM-1, or CD31) and von Willebrand factor immunocytochemistry. HMVECs were treated with increasing EMP doses, and then, the effects of EMPs on growth factor-induced proliferation and migration were tested. Proliferation was assessed by H-thymidine incorporation. EC migration was assayed by photographing microtubules of HMVECs and HUVECs in fibrin gel incubated with EMPs +/- growth factors for 48 h. The EMP effects on non-valve HUVECs were tested in parallel. EMPs inhibited HMVEC proliferation at high doses but stimulated HUVEC proliferation at all doses. In HMVECs, EMPs inhibited basic fibroblast growth factor- and vascular endothelial growth factor-induced proliferation and migration. Taken together, these data suggest EMPs regulate valvular EC proliferation in a dose-dependent manner and, furthermore, modulate growth factor signaling in ECs. These results implicate EMPs as a possible source of downstream EC dysfunction in disease states. EMPs may play a role in valvular leaflet injury in human disease by inhibiting normal growth and repair of endothelium.

Endothelium-derived microparticles induce endothelial dysfunction and acute lung injury.

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.

Rosiglitazone antagonizes vascular endothelial growth factor signaling and nuclear factor of activated T cells activation in cardiac valve endothelium.

Nuclear factor of activated T cells, Cytoplasmic 1 (NFATc1) is required for heart valve formation. Vascular endothelial growth factor (VEGF) signaling, mediated by NFATc1 activation, positively regulates growth of valvular endothelial cells. However, regulators of VEGF/NFATc1 signaling in valve endothelium are poorly understood. Peroxisome proliferator-activated receptor gamma (PPARgamma) inhibits NFATc1 activity in T cells and cardiomyocytes, but it is not known if PPARgamma controls NFATc1 function in endothelial cells. The authors hypothesize PPARgamma antagonizes VEGF signaling in valve endothelium by inhibiting NFATc1. Endothelial cells isolated from human valve leaflet tissue were shown by immunocytochemistry to express the endothelial-specific markers von Willebrand factor (vWF) and platelet endothelial cell adhesion molecule (PECAM)-1. VEGF-induced proliferation and migration of human pulmonary valve endothelial cells (HPVECs) were inhibited by rosiglitazone (ROSI), a specific ligand of PPARgamma activation, suggesting that PPARgamma disrupts VEGF signaling in the valve endothelium. ROSI also antagonized VEGF-mediated NFATc1 nuclear translocation in HPVECs, suggesting that PPARgamma inhibits VEGF signaling of NFATc1 activation in the valve. The effect of ROSI on nonvalve human umbilical vein endothelial cells (HUVECs) was tested in parallel and a similar inhibition of NFATc1 activation was observed. These data provide the first demonstration that ROSI negatively regulates VEGF signaling in the valve endothelium by a mechanism involving NFATc1 activation and nuclear translocation.

Requirement of RhoA activity for increased nuclear factor kappaB activity and PC-3 human prostate cancer cell invasion.

To determine the molecular mechanisms of aggressive prostate cancer behavior, we studied RhoGTPases in high and low invasive variants of PC-3 prostate cancer cells. Prior studies with these cells revealed that elevated nuclear factor kappaB (NF-kappaB) expression and activity were necessary for the highly invasive phenotype. In the current study, increased RhoA expression was found in the PC-3 highly invasive cells as compared with the PC-3 low invasive cells through cDNA array and Western blot analyses. Similarly, RhoA activity, as measured by the Rhotekin binding assay, was elevated in the PC-3 highly invasive cells. Transfection of these highly invasive cells with dominant negative RhoA N19 or treatment with 1.0 micro g/ml RhoA inhibitor C3 exoenzyme demonstrated that RhoA activity was necessary for both NF-kappaB activity and cellular invasion of a Matrigel reconstituted basement membrane. Furthermore, stable transfection of the PC-3 highly invasive cells with constitutively active RhoA Q63L resulted in activation of NF-kappaB activity and Matrigel invasion, effects reversed by treatment of the cells with C3 exoenzyme. RhoA was also shown to act through the motility component of the invasion process. RhoA activity was therefore both necessary and sufficient for the elevated NF-kappaB, invasion, and motility activities of the PC-3 highly invasive cells. These findings suggest molecular targets to control cancer cell invasion and aid in the development of definitive tools for predicting the invasive and metastatic potential of cancer cells.

Lipid-Free Apolipoprotein A-I Reduces Progression of Atherosclerosis by Mobilizing Microdomain Cholesterol and Attenuating the Number of CD131 Expressing Cells: Monitoring Cholesterol Homeostasis Using the Cellular Ester to Total Cholesterol Ratio.

BACKGROUND: Atherosclerosis is a chronic inflammatory disorder whose development is inversely correlated with high-density lipoprotein concentration. Current therapies involve pharmaceuticals that significantly elevate plasma high-density lipoprotein cholesterol concentrations. Our studies were conducted to investigate the effects of low-dose lipid-free apolipoprotein A-I (apoA-I) on chronic inflammation. The aims of these studies were to determine how subcutaneously injected lipid-free apoA-I reduces accumulation of lipid and immune cells within the aortic root of hypercholesterolemic mice without sustained elevations in plasma high-density lipoprotein cholesterol concentrations. METHODS AND RESULTS: Ldlr-/- and Ldlr-/- apoA-I-/- mice were fed a Western diet for a total of 12 weeks. After 6 weeks, a subset of mice from each group received subcutaneous injections of 200 µg of lipid-free human apoA-I 3 times a week, while the other subset received 200 µg of albumin, as a control. Mice treated with lipid-free apoA-I showed a decrease in cholesterol deposition and immune cell retention in the aortic root compared with albumin-treated mice, regardless of genotype. This reduction in atherosclerosis appeared to be directly related to a decrease in the number of CD131 expressing cells and the esterified cholesterol to total cholesterol content in several immune cell compartments. In addition, apoA-I treatment altered microdomain cholesterol composition that shifted CD131, the common ß subunit of the interleukin 3 receptor, from lipid raft to nonraft fractions of the plasma membrane. CONCLUSIONS: ApoA-I treatment reduced lipid and immune cell accumulation within the aortic root by systemically reducing microdomain cholesterol content in immune cells. These data suggest that lipid-free apoA-I mediates beneficial effects through attenuation of immune cell lipid raft cholesterol content, which affects numerous types of signal transduction pathways that rely on microdomain integrity for assembly and activation.

Requirement of cyclooxygenase-2 expression and prostaglandins for human prostate cancer cell invasion.

The PC-3 Low Invasive cells and the PC-3 High Invasive cells were used to investigate the correlation of the COX-2 expression and its arachidonic acid metabolites, prostaglandins, with their invasiveness through Matrigel using a Boyden chamber assay. The COX-2 expression in PC-3 High Invasive cells was approximately 3-fold higher than in PC-3 Low Invasive cells while the COX-1 expression was similar in both cell sublines. When incubated with arachidonic acid, PGE2 was the major prostaglandin produced by these cells. PC-3 High Invasive cells produced PGE2 approximately 2.5-fold higher than PC-3 Low Invasive cells. PGD2 was the second most abundant prostaglandin produced by these cells. Both indomethacin (a nonspecific COX inhibitor) and NS-398 (a specific COX-2 inhibitor) inhibited the production of prostaglandins and the cell invasion. PGE2 alone did not induce the cell invasion of PC-3 Low Invasive cells. However, PGE2 reversed the inhibition of cell invasion by NS-398 and enhanced the cell invasion of the PC-3 High Invasive cells. In contrast, PGD2 slightly inhibited the cell invasion. These results suggest that in the PC-3 Low Invasive cells, COX-2-derived PGE2 may not be sufficient to induce cell invasion while in the PC-3 High Invasive cells, PGE2 may be sufficient to act as an enhancer for the cell invasion. Further, PGD2 may represent a weak inhibitor and counteracts the effect of PGE2 in the cell invasion.

Clinical CVVH model removes endothelium-derived microparticles from circulation.

BACKGROUND: Endothelium-derived microparticles (EMPs) are submicron vesicles released from the plasma membrane of endothelial cells in response to injury, apoptosis or activation. We have previously demonstrated EMP-induced acute lung injury (ALI) in animal models and endothelial barrier dysfunction in vitro. Current treatment options for ALI are limited and consist of supportive therapies. We hypothesize that standard clinical continuous venovenous hemofiltration (CVVH) reduces serum EMP levels and may be adapted as a potential therapeutic intervention. MATERIALS AND METHODS: EMPs were generated from plasminogen activation inhibitor-1 (PAI-1)-stimulated human umbilical vein endothelial cells (HUVECs). Flow cytometric analysis was used to characterize EMPs as CD31- and annexin V-positive events in a submicron size gate. Enumeration was completed against a known concentration of latex beads. Ultimately, a concentration of ~650,000 EMP/mL perfusate fluid (total 470 mL) was circulated through a standard CVVH filter (pore size 200 µm, flow rate 250 mL/hr) for a period of 70 minutes. 0.5 mL aliquots were removed at 5- to 10-minute intervals for flow cytometric analysis. EMP concentration in the dialysate was measured at the end of 4 hours to better understand the fate of EMPs. RESULTS: A progressive decrease in circulating EMP concentration was noted using standard CVVH at 250 mL/hr (a clinical standard rate) from a 470 mL volume modelling a patient's circulation. A 50% reduction was noted within the first 30 minutes. EMPs entering the dialysate after 4 hours were 5.7% of the EMP original concentration. CONCLUSION: These data demonstrate that standard CVVH can remove EMPs from circulation in a circuit modelling a patient. An animal model of hemofiltration with induction of EMP release is required to test the therapeutic potential of this finding and potential of application in early treatment of ALI.

Innominate artery compression of trachea.

Innominate artery may cross the trachea and cause airway obstruction is a rare cause of vascular obstruction of airway. We describe a child with stridor, inability to extubate in whom the diagnosis was suspected on fluoroscopy and confirmed by angiography. Reimplantation of the innominate artery resulted in excellent results with longterm follow up. Variantions of this condition and modalities for confirmation of diagnosis are discussed.

Effects of D-4F on vasodilation, oxidative stress, angiostatin, myocardial inflammation, and angiogenic potential in tight-skin mice.

Systemic sclerosis (scleroderma, SSc) is an autoimmune, connective tissue disorder that is characterized by impaired vascular function, increased oxidative stress, inflammation of internal organs, and impaired angiogenesis. Tight skin mice (Tsk(-/+)) have a defect in fibrillin-1, resulting in replication of many of the myocardial and vascular features seen in humans with SSc. D-4F is an apolipoprotein A-I (apoA-I) mimetic that improves vascular function in diverse diseases such as hypercholesterolemia, influenza, and sickle cell disease. Tsk(-/+) mice were treated with either phosphate-buffered saline (PBS) or D-4F (1 mg.kg(-1).day(-1) for 6-8 wk). Acetylcholine and flow-induced vasodilation were examined in facialis arteries. Proinflammatory HDL (p-HDL) in murine and human plasma samples was determined by the cell-free assay. Angiostatin levels in murine and human plasma samples were determined by Western blot analysis. Hearts were examined for changes in angiostatin and autoantibodies against oxidized phosphotidylcholine (ox-PC). Angiogenic potential in thin sections of murine hearts was assessed by an in vitro vascular endothelial growth factor (VEGF)-induced endothelial cell (EC) tube formation assay. D-4F improved endothelium-, endothelial nitric oxide synthase-dependent, and flow-mediated vasodilation in Tsk(-/+) mice. Tsk(-/+) mice had higher plasma p-HDL and angiostatin levels than C57BL/6 mice, as did SSc patients compared with healthy control subjects. Tsk(-/+) mice also had higher triglycerides than C57BL/6 mice. D-4F reduced p-HDL, angiostatin, and triglycerides in the plasma of Tsk(-/+) mice. Tsk(-/+) hearts contained notably higher levels of angiostatin and autoantibodies against ox-PC than those of control hearts. D-4F ablated angiostatin in Tsk(-/+) hearts and reduced autoantibodies against ox-PC by >50% when compared with hearts from untreated Tsk(-/+) mice. Angiogenic potential in Tsk(-/+) hearts was increased only when the Tsk(-/+) mice were treated with D-4F (1 mg.kg(-1).day(-1), 6-8 wk), and cultured sections of hearts from the D-4F-treated Tsk(-/+) mice were incubated with D-4F (10 microg/ml, 5-7 days). Failure to treat the thin sections of hearts and Tsk(-/+) mice with D-4F resulted in loss of VEGF-induced EC tube formation. D-4F improves vascular function, decreases myocardial inflammation, and restores angiogenic potential in the hearts of Tsk(-/+) mice. As SSc patients have increased plasma p-HDL and angiostatin levels similar to the Tsk(-/+) mice, D-4F may be effective at treating vascular complications in patients with SSc.