Endothelial Adhesion Molecule Expression Research Articles

Pharmacological Tie2 activation in kidney transplantation.

AIMTo investigate the therapeutic potential of vasculotide (VT) - a Tie2 activating therapeutic - in kidney transplantation.METHODSWe performed a murine MHC-mismatched renal transplant model (C57Bl/6 male into Balb/c female) with 60 min cold and 30 min warm ischemia time. 500 ng VT was administered i.p. to donor mice 1 h before organ removal. In addition, recipients received 500 ng VT i.p. directly and 3 d after surgery. Survival was monitored and remaining animals were sacrificed 28 d after transplantation. In this model, we analyzed: (1) organ function; (2) Kaplan-Meier survival; (3) organ damage (periodic acid Schiff staining) via semi-quantitative scoring [0-4 (0 = no injury/inflammation to 4 = very severe injury/inflammation)]; (4) expression of renal endothelial adhesion molecules (ICAM-1) via immunofluorescence (IF) staining, immunoblotting and qPCR; (5) infiltration of inflammatory cells (IF Gr-1, F4/80); and (6) fibrosis via staining of α-smooth muscle actin (αSMA), Sirius red staining and immunoblotting of SMAD3 activation.RESULTSExogenous activation of Tie2 with VT resulted in diminished expression of peritubular and glomerular endothelial adhesion molecules. Consequently, infiltration of inflammatory cells (analyzed as ICAM-1, Gr-1 and F4/80 positive cells) was reduced in VT-treated mice compared to controls. Additionally, VT was protective against fibrogenesis after kidney transplantation. Trends towards lower serum creatinine (vehicle: 142 ± 17 μmol/L vs VT: 94 ± 23 μmol/L), urea (vehicle: 76 ± 5 mmol/L vs VT: 60 ± 8 mmol/L) and lactate dehydrogenase (vehicle: 1288 ± 383 iU vs VT: 870 ± 275 iU) were observed on day 6 after transplantation. Kaplan-Meier survival analysis showed improved survival rates in the VT-treated mice that did not reach statistical significance (27% vs 54%, P = 0.24, n = 11 per group). Exogenous activation of Tie2 via VT might reduce infiltration of inflammatory cells into renal tissue thereby protecting the transplant from early graft dysfunction potentially affecting long-term function.CONCLUSIONProtection of the endothelial microvasculature via the Tie2 axis in the early transplant setting might hold promise as a therapeutic target.

<i>Sphaeranthus indicus</i>: Traditional Wisdom to Modern Medicine—An Orally Active, Potent Cytokine Inhibitor for the Management of Inflammatory Disorders

Tumor necrosis factor (TNF-α) is a key regulator of the inflammatory and tissue destruc-tive pathways in rheumatoid arthritis (RA). The clinical success of anti-TNF-α and an-ti-IL-17 biologics has validated the concept that cytokine blockade is beneficial in RA. However, as these drugs are parenterally administered, our efforts are directed at identifying a novel orally active TNF-α inhibitor with a therapeutic profile similar to that of biologics. Since plants are natural immunomodulators, we explored the immunomodulatory potential of Sphaeranthus indicus extract. In our studies, the extract dose-dependently inhibited the release of cytokines in stimulated human peripheral blood mononuclear cells (hPBMCs), and their spontaneous release in synovial cells derived from patients suffering from RA. TNF-α and IFN-γ induced release of p40 subunit of IL-12/ IL-23, and p19 subunit of IL-23 in differentiated THP-1 cells is potently blocked. The expression of endothelial cell adhesion molecules in TNF-α-stimulated HUVECs was also potently inhibited. The oral treatment significantly and dose-dependently reduced LPS-induced TNF-α and IL-1β production in mice. Disease regression was seen in collagen-induced arthritis in DBA/1J mice, which was validated along with radiological and histopathological evaluation. Therefore, the extract of Sphaeranthus indicus could be used in the management of inflammatory conditions.

Activation of an endothelial Notch1-Jagged1 circuit induces VCAM1 expression, an effect amplified by interleukin-1β.

The Notch1 and Notch4 signaling pathways regulate endothelial cell homeostasis. Inflammatory cytokines induce the expression of endothelial adhesion molecules, including VCAM1, partly by downregulating Notch4 signaling. We investigated the role of endothelial Notch1 in this IL-1β-mediated process. Brief treatment with IL-1β upregulated endothelial VCAM1 and Notch ligand Jagged1. IL-1β decreased Notch1 mRNA levels, but levels of the active Notch1ICD protein remained constant. IL-1β-mediated VCAM1 induction was downregulated in endothelial cells subjected to pretreatment with a pharmacological inhibitor of the γ-secretase, which activates Notch receptors, producing NotchICD. It was also downregulated in cells in which Notch1 and/or Jagged1 were silenced.Conversely, the forced expression of Notch1ICD in naïve endothelial cells upregulated VCAM1 per se and amplified IL-1β-mediated VCAM1 induction. Jagged1 levels increased and Notch4 signaling was downregulated in parallel. Finally, Notch1ICD and Jagged1 expression was upregulated in the endothelium of the liver in a model of chronic liver inflammation.In conclusion, we describe here a cell-autonomous, pro-inflammatory endothelial Notch1-Jagged1 circuit (i) triggering the expression of VCAM1 even in the absence of inflammatory cytokines and (ii) enhancing the effects of IL-1β. Thus, IL-1β regulates Notch1 and Notch4 activity in opposite directions, consistent with a selective targeting of Notch1 in inflamed endothelium.

Co-Administration of Hydroxyurea and a Specific AKT2 Inhibitor Has Beneficial Effects on Acute Vaso-Occlusive Events and Survival in Sickle Cell Disease Mice

We recently demonstrated that the basal levels of AKT phosphorylation were significantly increased in neutrophils and platelets isolated from sickle cell disease (SCD) patients compared with those cells from healthy donors and that specific inhibition of AKT2 impaired neutrophil-endothelial cell and neutrophil-platelet interactions in venules of Berkeley (SCD) mice (Li et al. J Clin Invest 2014). Although hydroxyurea (HU), an inducer of fetal hemoglobin, is the main therapy for treatment of SCD, it is unclear whether the short-term treatment with HU has immediate benefits on acute vaso-occlusive events in SCD. Using real-time fluorescence intravital microscopy, we demonstrated that co-administration of HU (100 microg/g body weight, iv injection) and Akti XII (3 microg/g body weight, iv injection), an AKT2 inhibitor, efficiently reduced neutrophil adhesion and platelet-neutrophil aggregation in cremaster muscle venules of TNF-α-challenged Berkeley mice. Importantly, compared with HU or Akti XII treatment alone, treatment with both agents significantly improved blood flow rates and survival in the mice. Further, similar results were obtained in Berkeley mice challenged with hypoxia (8% oxygen for 3 hours) and subsequent reoxygenation (room air for 3 hours). As determined by histochemistry of cremaster muscle sections following intravital microscopy of Berkeley mice, the expression of endothelial E-selectin and intercellular adhesion molecule 1 (ICAM-1) was significantly decreased by treatment with either HU or Akti XII. Leukocyte transmigration in the lung section of Berkeley mice was also inhibited by either agent, and the inhibitory effect was potentiated by co-administration of HU and Akti XII. Using the plasma and isolated cells of Berkeley mice, we found that the level of plasma nitric oxide species (NOx) was significantly elevated by treatment with HU but not Akti XII and that AKT2 phosphorylation levels in activated neutrophils and platelets were reduced by treatment with Akti XII but not HU. Taken together, these results suggest that short-term treatment of Berkeley mice with either HU or Akti XII inhibits inflammatory conditions: treatment with HU significantly increases plasma NOx levels, treatment with Akti XII decreases AKT2 phosphorylation in neutrophils and platelets without affecting plasma NOx levels, and administration of either agent reduces the surface expression of ICAM-1 and E-selectin on activated endothelial cells. Thus, our results provide evidence that co-administration of HU and a specific AKT2 inhibitor has immediate benefits on acute vaso-occlusive events and survival in SCD mice exceeding those seen for single therapy. DisclosuresNo relevant conflicts of interest to declare.

Hydroxyurea with AKT2 inhibition decreases vaso-occlusive events in sickle cell disease mice

AbstractHeterotypic cell-cell adhesion and aggregation mediate vaso-occlusive events in patients with sickle cell disease (SCD). Although hydroxyurea (HU), an inducer of fetal hemoglobin, is the main therapy for treatment of SCD, it is unclear whether it has immediate benefits in acute vaso-occlusive events in SCD patients. Using real-time fluorescence intravital microscopy, we demonstrated that short-term coadministration of HU and Akti XII, an AKT2 inhibitor, efficiently reduced neutrophil adhesion and platelet-neutrophil aggregation in venules of Berkeley (SCD) mice challenged with tumor necrosis factor α (TNF-α) or hypoxia/reoxygenation. Importantly, compared with HU or Akti XII treatment alone, short-term treatment with both agents significantly improved survival in those mice. We found that the level of plasma nitric oxide species was elevated by HU but not Akti XII, AKT2 phosphorylation levels in activated neutrophils and platelets were reduced by Akti XII but not HU, and the expression of endothelial E-selectin and intercellular adhesion molecule 1 was decreased by either agent. Our results suggest that short-term coadministration of HU and Akti XII has immediate benefits for acute vaso-occlusive events and survival in SCD mice exceeding those seen for single therapy.

Abstract 15384: Laminar Flow Regulates Endothelial Cell Metabolism via Long Non-coding RNA lncflow2 (Best of Basic Science Abstract)

Considering that the transcriptome consists largely of non-coding transcripts, we analysed the impact of atheroprotective laminar flow (20 dynes/cm2) on the expression of long non-coding RNAs (lncRNA) in human endothelial cells (EC) by RNA deep sequencing. Among the significantly up-regulated lncRNAs, we specifically confirmed the up-regulation of lncflow2 upon laminar flow (3.4±1.5, p<0.05) and by overexpressing the flow-induced transcription factor KLF2 (1.95+0.2, p<0.05) by qRT-PCR. Lncflow2 is preferentially localized to the nucleus and RNA immunoprecipitation data indicate an association with the chromatin mark H3K27me3 suggesting that lncflow2 might epigenetically control gene expression. Functionally, the reduction of lncflow2 using LNA-Gapmers suppressed spheroid sprout formation of ECs (34.7 % reduction, p<0.05) and increased expression of endothelial adhesion molecules suggesting that lncflow2 improves EC function and reduces endothelial inflammation. Furthermore, gene ontology analysis of exon microarray analysis with RNA of lncflow2-silenced ECs revealed a significant regulation of metabolic pathways, such as the mevalonate pathway, indicated by elevated acetyl-CoA-acetyltransferase-2, HMG-CoA-reductase and HMG-CoA-synthase-1 expression (confirmed by RT-PCR; all p<0.05). In this context, inhibition of the HMG-Co-reductase by atorvastatin rescued the diminished spheroid sprouting upon loss of lncflow2. To elucidate additional effects on endothelial metabolic function, a mass spectrometry metabolome screening revealed significant alterations in amino acid, glucose and lipid metabolism. Seahorse analysis confirmed these data showing an elevated respiration and fatty acid oxidation in lncflow2 silenced ECs. Our data identify lncflow2 as a flow-regulated lncRNA that modifies EC metabolic responses by controlling metabolism-associated genes- particularly of the mevalonate pathway. Thereby, lncflow2 critically modifies EC functions and may play a regulating role in cellular metabolic adaption to stressors.

Abstract 12514: Elevated Intracellular Adenosine Epigenetically Regulates Endothelial Inflammation (Best of Basic Science Abstract)

Background and Objectives: Adenosine is a potent modulator of endothelial inflammation. However, the molecular mechanisms by which adenosine regulates endothelial inflammation has not been fully understood. This study aims to determine the effect of elevated intracellular adenosine on endothelial inflammation and investigate the involvement of epigenetic mechanism in this effect. Methods and Results: Adenosine but not adenosine receptor agonists alleviated the TNF-α-induced expression of endothelial adhesion molecules (E-selectin, ICAM-1 and VCAM-1). This effect was abrogated by nucleoside transporter inhibitor NBMPR but not by adenosine receptor antagonists. Knockdown of adenosine metabolic enzyme adenosine kinase (ADK) by shRNA or inhibition of ADK by inhibitors augmented intracellular adenosine and also attenuated TNF-α-induced expression of adhesion molecules. This effect was also not affected by adenosine receptor antagonists. Mechanistically, adenosine treatment or ADK knockdown deactivated the SAH hydrolase (SAHH) and histone H3K4 methyltransferase (HMT) and lowered H3K4 di- and tri- methylation levels. Erasing these histone marks by inhibition of SAHH and depletion of WDR5, a core subunit of HMT complex blocked the anti-inflammatory effects of adenosine and ADK knockdown. Co-IP assay showed that ADK can bind with SAHH and WDR5 to methylate H3K4 in a spatially effective manner. With the intravital microscopy we observed the compromised rolling and adhesion of leukocytes in postcapillary venues of endothelial specific ADK deficient mice. Furthermore, atheroprotection in Apoe-/- mice on a Western diet and neuroprotection in acute cerebral ischemic mice were conferred in endothelial specific ADK deficient mice. Conclusion: Our study provides evidence that intracellular adenosine attenuates endothelial inflammation through epigenetic pathways and ADK is a promising therapeutic target to treat inflammatory vascular diseases.

Pin1 inhibitor Juglone prevents diabetic vascular dysfunction

BackgroundAtherosclerosis is a major cause of mortality in patients with diabetes. However, novel breakthrough therapies have yet to be approved in this setting. Prolyl-isomerase-1 (Pin1) is emerging as a key molecule implicated in vascular oxidative stress and inflammation. In the present study, we investigate whether pharmacological inhibition of Pin1 may protect against diabetes-induced oxidative stress, endothelial dysfunction and vascular inflammation. Methods and ResultsExperiments were performed in human aortic endothelial cells (HAECs) exposed to normal (5mmol/L) or high glucose (25mmol/L) concentrations, in the presence of Pin1 inhibitor Juglone (10μM) or vehicle (<1% ethanol). In parallel, streptozotocin-induced diabetic mice were treated with Juglone i.p. every other day for 30days (1mg/Kg). Organ chamber experiments were performed in aortic rings to assess endothelium-dependent relaxations to acetylcholine (Ach 10−9 to 10−6mol/L). Mitochondrial oxidative stress, organelle integrity as well as NF-kB-dependent inflammatory signatures were determined both in HAECs and aortas from diabetic mice. In HAECs, ambient hyperglycemia increased mitochondrial superoxide anion generation while treatment with Juglone prevented this phenomenon. Pharmacological inhibition of Pin1 also preserved mitochondrial integrity, nitric oxide availability and endothelial expression of adhesion molecules. Interestingly enough, endothelial dysfunction, oxidative stress and NF-kB-driven inflammation were significantly attenuated in diabetic mice chronically treated with Juglone as compared to vehicle-treated animals. ConclusionPharmacological inhibition of Pin1 by Juglone prevents hyperglycemia-induced vascular dysfunction. Taken together, our findings may set the stage for novel therapeutic approaches to prevent vascular complications in patients with diabetes.

Glossogyne tenuifolia Extract Inhibits TNF-α-Induced Expression of Adhesion Molecules in Human Umbilical Vein Endothelial Cells via Blocking the NF-kB Signaling Pathway.

Chronic inflammation plays a pivotal role in the development of atherosclerosis, where the pro-inflammatory cytokine-induced expression of endothelial adhesion molecules and the recruitment of monocytes are the crucial events leading to its pathogenesis. Glossogyne tenuifolia ethanol extract (GTE) is shown to have potent anti-inflammatory and antioxidant activities. We evaluated the effects of GTE and its major components, luteolin (lut), luteolin-7-glucoside (lut-7-g), and oleanolic acid (OA) on TNF-α-induced expression of adhesion molecules in human umbilical vein endothelial cells (HUVECs). The results demonstrated that GTE, lut, and lut-7-g attenuated the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in TNF-α-activated HUVECs, and inhibited the adhesion of monocytes to TNF-α-activated HUVECs. The TNF-α-induced mRNA expression of ICAM-1 and VCAM-1 was also suppressed, revealing their inhibitory effects at the transcriptional level. Furthermore, GTE, lut, and lut-7-g blocked the TNF-α-induced degradation of nuclear factor-κB inhibitor (IκB), an indicator of the activation of nuclear factor-κB (NF-κB). In summary, GTE and its bioactive components were effective in preventing the adhesion of monocytes to cytokine-activated endothelium by the inhibition of expression of adhesion molecules, which in turn is mediated through blocking the activation and nuclear translocation of NF-κB. The current results reveal the therapeutic potential of GTE in atherosclerosis.

Imaging Inflammation in Cerebrovascular Disease.

Imaging inflammation in large intracranial artery pathology may play an important role in the diagnosis of and risk stratification for a variety of cerebrovascular diseases. Looking beyond the lumen has already generated widespread excitement in the stroke community, and the potential to unveil molecular processes in the vessel wall is a natural evolution to develop a more comprehensive understanding of the pathogenesis of diseases, such as ICAD and brain aneurysms.

Evaluation of cytokines and soluble adhesion molecules in patients with newly diagnosed acute myeloid leukemia: the role of TNF-alpha and FLT3-ITD.

Acute myeloid leukemia (AML) cells are highly resistant to therapy. The presumed molecular basis of this resistance is the effect of tumor necrosis factor alpha (TNF-α) and other cytokines on endothelial adhesion molecule expression. The aim of this study was to test the hypothesis that cytokines and soluble adhesion molecules correlate in AML. Baseline serum levels of 17 cytokines and 5 soluble adhesion molecules were measured in 53 AML patients using biochip array technology. Age, leukocyte count, secondary AML, CRP, FLT3-ITD and remission were variables. Statistical analysis was performed in R version 3.1.2. VCAM-1 correlated with ICAM-1 (P < 0.0001), E-selectin (P < 0.0001), leukocyte count (P = 0.0005) and TNF-α (P = 0.0035). E-selectin correlated with leukocyte count (P < 0.0001), P-selectin (P = 0.0032) and MCP-1 (P = 0.0119). CRP correlated with IL-6 (P < 0.0001), leukocyte count negatively correlated with IL-7 (P = 0.0318). FLT3-ITD was associated with higher E-selectin (P = 0.0010) and lower IL-7 (P = 0.0252). Secondary AML patients were older. Failure of induction therapy was associated with significantly higher CRP and lower P-selectin. Leukocyte count (P < 0.0001), FLT3-ITD (P = 0.0017) and secondary AML (P = 0.0439) influenced the principal component. Leukemic cells can modulate the microenvironment. Cytokine, adhesion molecule levels and leukocyte count correlate in AML. Understanding these mechanisms may form the basis of novel therapeutic approaches.

Endothelial Dysfunction and Altered Coagulation As Mediators of Thromboembolism in Behçet Disease.

Behçet disease (BD) is a rare multisystem, inflammatory disease of unknown etiology with vascular involvement and associated thrombogenicity. This review aims to describe the involvement of various mediators in endothelial cell damage and in the hypercoagulable state of BD. The scenario of the chronic inflammation present in BD shows an increased oxidative condition that contributes to endothelial cell damage and induces platelet, leukocyte, and endothelial cell activation through the release of proinflammatory cytokines and chemokines. These factors, together with the increased levels of homocysteine observed in BD patients, induce the endothelial cell expression of adhesion molecules (VCAM-1 and ICAM-1) and tissue factor; the release of cytokines, soluble CD40L (sCD40L), matrix metalloproteinase-9, and blood coagulation factor V; the inhibition of fibrinolysis; the disruption of nitric oxide metabolism; and the increase in platelet reactivity and lipid peroxidation. Endothelial cell dysfunction leads to a prothrombotic and antifibrinolytic phenotype in BD patients. Increased levels of homocysteine, fibrinogen, and plasminogen activator inhibitor type 1 seem to be involved in the procoagulant condition of this pathology that has been verified by end-point tests as well as by global coagulation tests.

Degradable poly(apigenin) polymer inhibits tumor cell adhesion to vascular endothelial cells.

Cancer and the inflammatory system share a complex intertwined relationship. For instance, in response to an injury or stress, vascular endothelial cells will express cell adhesion molecules as a means of recruiting leukocytes. However, circulating tumor cells (CTCs) have been shown to highjack this expression for the adhesion and invasion during the metastatic cascade. As such, the initiation of endothelial cell inflammation, either by surgical procedures (cancer resection) or chemotherapy can inadvertently increase the metastatic potential of CTCs. Yet, systemic delivery of anti-inflammatories, which weaken the entire immune system, may not be preferred in some treatment settings. In this work, we demonstrate that a long-term releasing flavone-based polymer and subsequent nanoparticle delivery system can inhibit tumor cell adhesion, through the suppression of endothelial cell adhesion molecule expression. The degradation of a this anti-inflammatory polymer provides longer term, localized release profile of active therapeutic drug in nanoparticle form as compared with that of the free drug, permitting more targeted anti-metastatic therapies. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1438-1447, 2016.

Acute hemolytic vascular inflammatory processes are prevented by nitric oxide replacement or a single dose of hydroxyurea

AbstractHemolysis and consequent release of cell-free hemoglobin (CFHb) impair vascular nitric oxide (NO) bioavailability and cause oxidative and inflammatory processes. Hydroxyurea (HU), a common therapy for sickle cell disease (SCD), induces fetal Hb production and can act as an NO donor. We evaluated the acute inflammatory effects of intravenous water-induced hemolysis in C57BL/6 mice and determined the abilities of an NO donor, diethylamine NONOate (DEANO), and a single dose of HU to modulate this inflammation. Intravenous water induced acute hemolysis in C57BL/6 mice, attaining plasma Hb levels comparable to those observed in chimeric SCD mice. This hemolysis resulted in significant and rapid systemic inflammation and vascular leukocyte recruitment within 15 minutes, accompanied by NO metabolite generation. Administration of another potent NO scavenger (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) to C57BL/6 mice induced similar alterations in leukocyte recruitment, whereas hemin-induced inflammation occurred over a longer time frame. Importantly, the acute inflammatory effects of water-induced hemolysis were abolished by the simultaneous administration of DEANO or HU, without altering CFHb, in an NO pathway–mediated manner. In vitro, HU partially reversed the Hb-mediated induction of endothelial proinflammatory cytokine secretion and adhesion molecule expression. In summary, pathophysiological levels of hemolysis trigger an immediate inflammatory response, possibly mediated by vascular NO consumption. HU presents beneficial anti-inflammatory effects by inhibiting rapid-onset hemolytic inflammation via an NO-dependent mechanism, independently of fetal Hb elevation. Data provide novel insights into mechanisms of hemolytic inflammation and further support perspectives for the use of HU as an acute treatment for SCD and other hemolytic disorders.

A critical role for ATF2 transcription factor in the regulation of E-selectin expression in response to non-endotoxin components of Neisseria meningitidis.

SummaryVascular injury is a serious complication of sepsis due to the gram‐negative bacterium Neisseria meningitidis. One of the critical early steps in initiating this injury is via the interaction of leucocytes, particularly neutrophils, with adhesion molecules expressed on inflamed endothelium. We have previously demonstrated that both lipopolysaccharide (LPS) and non‐LPS components of meningococci can induce very high levels of expression of the vascular endothelial cell adhesion molecule E‐selectin, which is critical for early tethering and capture of neutrophils onto endothelium under flow. Using an LPS‐deficient strain of meningococcus, we showed that very high levels of expression can be induced in primary endothelial cells, even in the context of weak activation of the major host signal transduction factor [nuclear factor‐κB (NF‐κB)]. In this study, we show that the particular propensity for N . meningitidis to induce high levels of expression is regulated at a transcriptional level, and demonstrate a significant role for phosphorylation of the ATF2 transcription factor, likely via mitogen‐activated protein (MAP) kinases, on the activity of the E‐selectin promoter. Furthermore, inhibition of E‐selectin expression in response to the lpxA− strain by a p38 inhibitor indicates a significant role of a p38‐dependent MAPK signalling pathway in ATF2 activation. Collectively, these data highlight the role that LPS and other bacterial components have in modulating endothelial function and their involvement in the pathogenesis of meningococcal sepsis. Better understanding of these multiple mechanisms induced by complex stimuli such as bacteria, and the specific inflammatory pathways they activate, may lead to improved, focused interventions in both meningococcal and potentially bacterial sepsis more generally.

Excessive dietary linoleic acid induces proinflammatory markers in rats

Following the historical dietary recommendations, the substitution of polyunsaturated fatty acids (PUFAs) for saturated fatty acids (SFAs) resulted in a dramatic increase of linoleic acid (LA) in the Western diet. While proatherogenic properties of SFAs have been described, the involvement of LA on the inflammatory process remains controversial. Herein, we evaluated the effects of an excessive LA intake on the cytokine-induced expression of endothelial adhesion molecules vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1), through the nuclear factor (NF)-κB pathway, in comparison with a control diet and regarding a “positive” SFA diet. Wistar rats were fed experimental diets — a control diet or diets enriched with LA or SFA — for 11 weeks. Plasma lipid parameters and proinflammatory cytokine production such as interleukin-1β and tumor necrosis factor (TNF)-α were analyzed. Expression of endothelial adhesion molecules and NF-κB was determined by immunohistochemical analysis. No difference was observed in body weight. The enriched diets did not affect triglyceride and total cholesterol levels in plasma. Our results demonstrated that excessive dietary LA intake increased TNF-α levels (P<.05) in plasma. Rats fed the LA-enriched diet showed a significantly higher expression of VCAM-1, ICAM-1 and NF-κB in aortas. In addition, our results demonstrated that an excess of LA is more efficient to activate endothelial molecular process than an excess of SFA. The present study provides further support for the proinflammatory properties of LA and suggests an LA-derivatives pathway involved in the inflammatory process.

Intravenous infusion of haptoglobin for the prevention of adverse clinical outcome in Sickle Cell Disease

Sickle Cell Disease (SCD) is a genetic condition which manifests as altered hemoglobin (Hb) protein that can aggregate under hypoxic conditions. The resultant sickled erythrocytes experience premature hemolysis, releasing an estimated 10g of free Hb (fHb) into the intravascular space. FHb participates in redox reactions creating various reactive oxygen species which rapidly and irreversibly scavenge nitric oxide, thereby attenuating its vasodilatory, antithrombotic, and anti-inflammatory properties. FHb also induces endothelial expression of adhesion molecules, triggering leukocyte margination at the vessel wall. These mechanisms participate in diverse SCD-associated clinical events including nephropathy, pulmonary hypertension, chronic leg ulceration, and ischemic events. FHb also exerts a direct reno-toxic effect contributing to albuminuria which is an early, frequent manifestation of glomerular injury.Under normal conditions, fHb is effectively scavenged by the Hb-scavenging mechanism (HSM); this involves binding to haptoglobin (Hp), uptake via the Hb-scavenging receptor (CD163) on monocytes and metabolism by heme-oxygenase-1. This culminates in increased CD163 expression and release of anti-inflammatory by-products e.g. interleukin-10 (IL-10).In SCD, the Hb-binding capacity is overwhelmed by chronic hemolysis; our previous research shows serum Hp as the depleted component. This deficiency could result in the harmful consequences of circulating fHb going unbridled.The hypothesis we explore here is that Hp infusions, in excess of fHb concentration, will allow the HSM to remain functional, and thereby achieve improved clinical outcomes, tracking albuminuria as a sentinel. Albuminuria was selected because of its high prevalence in SCD and its relative ease of diagnosis and monitoring.The hypothesis may be evaluated in four phases: Phase 1 will determine the concentration of Hp needed to trigger the HSM as measured by induction of CD163 and IL-10 and the recovery of hemopexin. Phase 2 will investigate the half-life of HSM induction by analyzing the time-course of CD163 expression and IL-10 and hemopexin serum concentration. Phase 3 will determine patient eligibility for therapy, whether as treatment or prevention. Phase 4 will test the efficacy of Hp transfusions in a randomized control trial as measured by correction of albuminuria.Angiotensin converting enzyme inhibitors (ACEi) are currently the first-line treatment for SCD nephropathy, however hyperkalemia limits its use. Hydroxyurea, which has therapeutic value in many SCD adverse events, has yielded inconsistent effects on albuminuria. We are proposing the addition of an intervention more proximal in the hemolytic cascade. Boosting the exhausted Hb-scavenging capacity via Hp replacement therapy has the potential to modify multiple downstream clinical events.

Propofol ameliorates endothelial inflammation induced by hypoxia/reoxygenation in human umbilical vein endothelial cells: Role of phosphatase A2

Hypoxia/reoxygenation (H/R) induces endothelial inflammation with augmentation of endothelial adhesion molecules over-expression. Propofol was reported to attenuate endothelial adhesion molecule expression in some situations. Here, we examined the molecular mechanism for how propofol restored H/R-mediated up-regulation of endothelial adhesion molecules in human umbilical vein endothelial cells (HUVECs). Compared with the control group, H/R up-regulated expression of Pin-1 and PP2A, increased p66Shc–Ser36 phosphorylation, induced p66Shc mitochondrial translocation, O2− accumulation and NF-κB activation, and decreased eNOS–Ser1177 phosphorylation and nitric oxide (NO) production, thus up-regulating expression of endothelial adhesion molecules and increasing mononuclear-endothelial interaction. More importantly, except that propofol had no effect on H/R-induced p66Shc–Ser36 phosphorylation, most of H/R-mediated changes were alleviated by propofol, resulting in the reduction of endothelial adhesion molecules expression and mononuclear-endothelial adhesion. Moreover, we demonstrated the protective effect of propofol on H/R-induced endothelial inflammation was similar to that of calyculin A, an inhibitor of PP2A. In contrast, FTY720, an activator of PP2A, antagonized the effect of propofol. Our data indicated that propofol down-regulated PP2A expression, leading to reduced dephosphorylation of p66Shc–Ser36 and eNOS–Ser1177, which is associated with ROS accumulation and NO reduction, resulting in inhibition of endothelial adhesion molecule expression and mononuclear-endothelial interaction.

Abstract 47: High-Density Lipoproteins Induce Neutrophil-miRNA Transcription and Export: Contribution to HDL-miRNA Cell to Cell Communication

Background: We have recently shown that high-density lipoproteins (HDL)-associated miR-223 is exported from HDL to endothelial cells in a key pathway mechanism by which HDL mediate the suppression of endothelial cell adhesion molecule expression. Interestingly, miR-223 was found to be the most abundant miRNA in polymorphonuclear neutrophils (PMNs) and miR-223 is critical to PMNs activation. Here, we sought to determine if PMNs export miR-223 to HDL and if PMNs are a significant contributor to HDL-miR-223 cell-to-cell communication, which we previously reported. Results: To determine if PMNs export miR-223 to native HDL, PMNs and HDL were collected from 8 healthy donors by polymorph preps and density-gradient ultracentrifugation; respectively. HDL (1 mg/mL) were incubated with PMNs for 4 h and HDL were then isolated from culture media using apoA-I immunoprecipitation columns. Total RNA was isolated from HDL, and miR-223 levels were measured by TaqMan assays. miRNA export assays showed a significant increase in miR-223 on HDL post-PMN incubation compared to pre-incubation. To determine if other myeloid cells also export miR-223 to HDL, we isolated human monocytes by elutriation and differentiated them to macrophages (HMDM) over 7 days. Similar to PMNs, HMDM were also found to export miR-223 to HDL after 16 h. As a consequence of miR-223 export, we predicted that PMN cellular levels of mature miR-223 would decrease. Strikingly, we found no differences in cellular mature miR-223 levels in PMNs or HMDM treated with HDL. To determine if cellular miR-223 levels are supported by increased miR-223 transcription in response to HDL induction and miRNA export, we quantified primary (pri)-miR-223 levels using real-time PCR and TaqMan assays. We found that HDL induce a significant 3.75-fold increase in PMN pri-miR-223 levels after 4 h. Likewise; we found a significant 2.24-fold increase in HMDM pri-miR-223 levels after 16 h of HDL treatment Conclusion: PMNs export miR-223 to HDL; however, miRNA export does not reduce cellular mature miR-223 levels likely due to HDL-induced pri-miR-223 transcription. This suggests that HDL induce the transcription of miRNAs selected for export and indicates that cells can satisfy demand for export and maintain cellular miRNA levels.

Post-transcriptional control of Amblyomin-X on secretion of vascular endothelial growth factor and expression of adhesion molecules in endothelial cells.

Angiogenesis is a pivotal process of homeostasis and tissue repair, but it also favours neovascularisation syndromes and cancer nutrition. The chemical mediation of angiogenesis is complex, involving a balance between serine proteases and their inhibitors. We addressed the mechanisms of action of a Kunitz serine protease inhibitor (KPI) on spontaneous angiogenesis, using Amblyomin-X, a KPI designed from the cDNA library of the Amblyomma cajennense tick. Amblyomin-X treatment (10-1000ng/10μL; each 48h; 3 times) reduced the number of vessels in the subcutaneous dorsal tissue of male Swiss mice, as measured by intravital microscopy, haematoxylin-eosin staining, and PECAM-1 immunofluorescence labeling. Incubation of Amblyomin-X with t-End endothelial cells, a murine endothelial microvascular lineage, did not alter cell proliferation, cell-cycle phases, necrosis and apoptosis, and the production of nitric oxide and prostaglandin E2. Nevertheless, Amblyomin-X treatment reduced t-End migration and adhesion to Matrigel(®), and inhibited the VEGF-A secretion and VCAM-1 and β3 integrin expressions by posttranscriptional pathways. Together, data herein outline novel posttranscriptional mechanisms of KPIs on endothelial cells during angiogenesis and point out the possible application of Amblyomin-X as a local inhibitor to undesired neovascularisation process.