Plasma Levels Of sVCAM-1 Research Articles

Diabetic Endothelial Cell Glycogen Synthase Kinase 3β Activation Induces VCAM1 Ectodomain Shedding.

Soluble cell adhesion molecules (sCAMs) are secreted ectodomain fragments of surface adhesion molecules, ICAM1 and VCAM1. sCAMs have diverse immune functions beyond their primary function, impacting immune cell recruitment and activation. Elevated sVCAM1 levels have been found to be associated with poor cardiovascular disease (CVD) outcomes, supporting VCAM1's role as a potential diagnostic marker and therapeutic target. Inhibiting sVCAM1's release or its interaction with immune cells could offer cardioprotection in conditions such as diabetes. Membrane-bound surface adhesion molecules are widely expressed in a wide variety of cell types with higher expression in endothelial cells (ECs). Still, the source of sCAMs in the circulation is not clear. Hypothesizing that endothelial cells (ECs) could be a potential source of sCAMs, this study investigated whether dysfunctional EC signaling mechanisms during diabetes cause VCAM1 ectodomain shedding. Our results from samples from an inducible diabetic mouse model revealed increased sVCAM1 plasma levels in diabetes. Protein analysis indicated upregulated VCAM1 expression and metalloproteases ADAM10 and ADAM17 in diabetic ECs. ADAMs are known for proteolytic cleavage of adhesion molecules, contributing to inflammation. GSK3β, implicated in EC VCAM1 expression, was found to be activated in diabetic ECs. GSK3β activation in control ECs increased ADAM10/17 and VCAM1. A GSK3β inhibitor reduced active GSK3β and VCAM1 ectodomain shedding. These findings suggest diabetic ECs with elevated GSK3β activity led to VCAM1 upregulation and ADAM10/17-mediated sVCAM1 shedding. This mechanism underscores the potential therapeutic role of GSK3β inhibition in reducing the levels of circulating sVCAM1. The complex roles of sCAMs extend well beyond CVD. Thus, unraveling the intricate involvement of sCAMs in the initiation and progression of vascular disease, particularly in diabetes, holds significant therapeutic potential.

Soluble Vascular Cell Adhesion Molecule-1 as an Inflammation-Related Biomarker of Coronary Slow Flow.

Background: Coronary slow flow (CSF) is an angiographic entity characterized by delayed coronary opacification with no evident obstructive lesion in the epicardial coronary artery. Several studies have shown that the occurrence and development of CSF may be closely related to inflammation. Soluble vascular cell adhesion molecule-1 (sVCAM-1) is a biomarker related to inflammation. The aim of this study was to evaluate the correlation between plasma soluble VCAM-1 level and CSF occurrence and thus the predictive value of VCAM-1 for CSF. Methods: Forty-six CSF patients and thirty control subjects were enrolled. Corrected thrombolysis in myocardial infarction frame count (cTFC) was used to diagnose CSF. Functional status and quality of life were determined by the Seattle Angina Questionnaire (SAQ). Echocardiography was used to evaluate the systolic and diastolic function of the left ventricle (LV) and right ventricle (RV). The plasma levels of sVCAM-1, IL-6, and TNF-α were quantified by enzyme-linked immunosorbent assay. Results: Compared with the control group, the physical limitation score by the SAQ, the LV global longitudinal strain (GLS), mitral E, and mitral E/A decreased in patients with CSF, while the plasma IL-6 and TNF-α levels increased. The plasma sVCAM-1 level in the CSF group was significantly higher than that in the control group (186.03 ± 83.21 vs. 82.43 ± 42.12 ng/mL, p < 0.001), positively correlated with mean cTFC (r = 0.57, p < 0.001), and negatively correlated with the physical limitation score (r = −0.32, p = 0.004). Logistic regression analyses confirmed that plasma sVCAM-1 level (OR = 1.07, 95%CI: 1.03−1.11) is an independent predictor of CSF, and the receiver operating characteristic curve analysis showed that plasma sVCAM-1 levels had statistical significance in predicting CSF (area under curve = 0.88, p < 0.001). When the sVCAM-1 level was higher than 111.57 ng/mL, the sensitivity for predicting CSF was 87% and the specificity was 73%. Conclusions: Plasma sVCAM-1 level can be used to predict CSF and was associated with the clinical symptoms of patients. It may serve as a potential biomarker for CSF in the future.

SVCAM-1, and TGFβ1 in chronic phase, chronic myeloid leukemia patients treated with tyrosine kinase inhibitors

The outcome for chronic phase (CP) chronic myelogenous leukemia (CML) patients has changed dramatically since the introduction of tyrosine kinase inhibitor (TKI) therapy. We examined the characteristics of CML patients during TKI therapy by determining the plasma concentrations of soluble vascular cell adhesion molecule 1 (sVCAM-1), and transforming growth factor (TGFβ1) biomarkers. The plasma levels of sVCAM-1 and TGFβ1 were measured by ELISA at baseline and after 3 months of TKI treatment. The levels of sVCAM-1, and TGFβ1 were significantly elevated in patients with CML (P< 0.01). Dasatinib treatment was associated with a significant reduction in the levels of these biomarkers (P< 0.01). In conclusion, plasma levels of sVCAM-1 and TGFβ1 could have a role in the pathogenesis of CML and may be used as predictors of hematological and molecular responses to TKIs. A favorable outcome for Dasatinib therapy was observed.

Specific Features of the Coagulopathy Signature in Severe COVID-19 Pneumonia.

Rationale: COVID-19 displays distinct characteristics that suggest a unique pathogenesis. The objective of this study was to compare biomarkers of coagulopathy and outcomes in COVID-19 and non-COVID-19 patients with severe pneumonia.Methods: Thirty-six non-COVID-19 and 27 COVID-19 non-immunocompromised patients with severe pneumonia were prospectively enrolled, most requiring intensive care. Clinical and biological characteristics (including plasma biomarkers of coagulopathy) were compared.Results: At similar baseline severity, COVID-19 patients required mechanical ventilation (MV) for significantly longer than non-COVID-19 patients (p = 0.0049) and more frequently developed venous thrombotic complications (p = 0.031). COVID-19 patients had significantly higher plasma concentrations of soluble VCAM1 (sVCAM1) (5,739 ± 3,293 vs. 3,700 ± 2,124 ng/ml; p = 0.009), but lower levels of D-dimers, vWF-A2, sICAM1, sTREM1, VEGF, and P-selectin, compared to non-COVID-19 patients. Principal component analysis identified two main patterns, with a clear distinction between non-COVID-19 and COVID-19 patients. Multivariable regression analysis confirmed that sVCAM1 rising levels were independently associated with a longer duration of MV. Finally, we identified close correlations between sVCAM1 and some features of COVID-19 immune dysregulation (ie. CXCL10, GM-CSF, and IL-10).Conclusion: We identified specific features of the coagulopathy signature in severe COVID-19 patients, with higher plasma sVCAM1 levels, that were independently associated with the longer duration of mechanical ventilation.Clinical Trial Registration:ClinicalTrials.gov, identifier: NCT03505281.

Effects of a synbiotic on symptoms, and daily functioning in attention deficit hyperactivity disorder – A double-blind randomized controlled trial

Some prebiotics and probiotics have been proposed to improve psychiatric symptoms in children with autism. However, few studies were placebo-controlled, and there is no study on persons with an attention deficit hyperactivity disorder (ADHD) diagnosis. Our aim was to study effects of Synbiotic 2000 on psychiatric symptoms and functioning in children and adults with ADHD without an autism diagnosis. Children and adults (n = 182) with an ADHD diagnosis completed the nine weeks randomized double-blind parallel placebo-controlled trial examining effects of Synbiotic 2000 on the primary endpoints ADHD symptoms, autism symptoms and daily functioning, and the secondary endpoint emotion regulation, measured using the questionnaires SNAP-IV, ASRS, WFIRS, SCQ, AQ and DERS-16. Levels at baseline of plasma C-reactive protein and soluble vascular cell adhesion molecule-1 (sVCAM-1), central to leukocyte-endothelial cell adhesion facilitating inflammatory responses in tissues, were measured using Meso Scale Discovery. Synbiotic 2000 and placebo improved ADHD symptoms equally well, and neither active treatment nor placebo had any statistically significant effect on functioning or sub-diagnostic autism symptoms. However, Synbiotic 2000, specifically, reduced sub-diagnostic autism symptoms in the domain restricted, repetitive and stereotyped behaviors in children, and improved emotion regulation in the domain of goal-directed behavior in adults. In children with elevated sVCAM-1 levels at baseline as well as in children without ADHD medication, Synbiotic 2000 reduced both the total score of autism symptoms, and the restricted, repetitive and stereotyped behaviors. In adults with elevated sVCAM-1 at baseline, Synbiotic 2000 significantly improved emotion regulation, both the total score and four of the five subdomains. To conclude, while no definite Synbiotic 2000-specific effect was detected, the analysis of those with elevated plasma sVCAM-1 levels proposed a reduction of autism symptoms in children and an improvement of emotion regulation in adults with ADHD.Trial registration number: ISRCTN57795429.

Daily watermelon consumption decreases plasma sVCAM-1 levels in overweight and obese postmenopausal women

Postmenopausal status is associated with an increase in total and abdominal body fat as well as increased incidence of insulin resistance and cardiovascular disease. The purpose of this study was to determine if watermelon supplementation affects select systemic markers of atherosclerosis and measures of insulin resistance in overweight and obese postmenopausal women. We hypothesized that overweight and obese postmenopausal women consuming 100% watermelon puree daily for 6 weeks would have improved levels of select systemic markers connected with cardiovascular disease without changing markers of insulin resistance. To test this hypothesis, overweight and obese postmenopausal women were recruited to participate in this study. Participants were randomly assigned to either the control group (no intervention) or the watermelon puree group (WM) for 6 weeks. Plasma concentration of markers connected with atherosclerosis and glycemic control were measured pre- and poststudy. A significant 6% decrease in soluble vascular cell adhesion molecule-1 occurred pre- to poststudy in WM, P = .003. The pattern of change in fasting blood glucose (P = .633), insulin (P = .158), and homeostatic model assessment–estimated insulin resistance (P = .174) did not differ between groups. Pre- to poststudy increases were measured in the fasting plasma concentration of l-arginine (8%, P = .005), cis-lycopene (32%, P = .003), and trans-lycopene (42%, P = .003) in WM. We conclude that 6 weeks of watermelon supplementation improved soluble vascular cell adhesion molecule-1 levels, a marker connected to atherogenesis, independent of changes in body composition or glycemic control.

The VLA-4 Integrin Is Constitutively Activated in a Fraction of CD49d-Expressing Chronic Lymphocytic Leukemia Via Autonomous BCR-Mediated Signaling

Introduction. In chronic lymphocytic leukemia (CLL), CD49d, the alpha chain of the heterodimer CD49d/CD29 (VLA-4), is a strong negative prognosticator, and a key player of CLL microenvironmental interactions. The adhesive properties of VLA-4 can be rapidly inside-out activated by signals through the B-cell receptor (BCR), thus favoring the capability of the integrin to interact with its ligands. Especially, a continuous BCR signaling, which is either induced by canonical autoantigen-dependent mechanism, or by an autonomous manner, may augment VLA-4 activation. Aim. To investigate the constitutive VLA-4 activation state in CLL cells and to connect this feature with the presence of signals from the BCR. Methods. Constitutive VLA-4 activation was determined in flow cytometry by combining expression of CD49d and activated CD29, the latter using the conformation-sensitive anti-CD29 mAb HUTS21 (Tissino et al, J Exp Med, 2018) in whole blood (WB), as source of VLA-4 ligands, from: i) 1,044 consecutive CLL all with IGHV gene mutations available; ii) sequential samples (0, 14, 30, 90 days) from CLL patients treated in vivo with ibrutinib (IB) in real-world (n=15) and from a clinical trial (NCT02827617, n=15). HUTS21 staining was also performed in the presence of: i) plasma depletion/replacement; ii) soluble (s) VCAM-1; iii) fibronectin (FN); iv) blocking anti-CD49d (HP1/2) mAbs. ELISA assays were used to quantify sVCAM-1 in plasma samples (n=122). Antigene driven BCR signaling and autonomous BCR signaling were investigated by Ca++ influx assay in a 4-hydroxy tamoxifen (4-OHT) inducible manner in murine TKO cells system as described before (Dühren-von Minden M et al, Nature, 2012). VLA-4 activation was assessed by "real-time" flow cytometry measuring the binding of the VLA-4 ligand LDV-FITC and its replacement by unlabeled LDV. The calculated Koff values indicate the VLA-4 affinity state (Koff &amp;lt;0.02 s−1 high affinity; Koff &amp;gt;0.06 s−1 low affinity). BCR engagement was performed using goat F(ab′)2 anti-human IgM. Results. 1) A fraction of CD49d+ CLL shows constitutive VLA-4 activation - Out of 1,044 CLL, 534 (51%) expressed CD49d (cutoff 30%), and HUTS21 (cutoff 20%) was scored positive in 112/534 (21%) cases. HUTS21 staining was: i) impaired by depletion of plasma from WB samples (Fig.A), and reconstituted by specific plasma components (sVCAM1, FN; Fig.B); ii) impaired by pre-incubation with anti-CD49d HP2/1 blocking mAbs before addition of plasma, sVCAM-1 and FN (Fig.C). 2) Plasma levels of sVCAM-1 are dependent on the VLA-4 activation state - sVCAM-1 (n=122 plasma samples) was higher in CD49d+ vs CD49d- cases (p&amp;lt;0.0001); among CD49d+ cases, sVCAM-1 was lower in HUTS21+ cases (p=0.0096), suggesting ligand sequestration by activated surface VLA-4 (Fig.D). 3) Constitutive VLA-4 activation associates to specific BCR - By comparing cases expressing high vs low levels of constitutively activated VLA-4 (HUTS21&amp;gt;50% vs HUTS21&amp;lt;5%), a skewing in IGHV gene family usage was detected (Fig.E). In particular, cases bearing the IGHV gene families 4-34, 3-21, 4-39, 3-7 frequently associated with a high constitutive VLA-4 activation state (12/26 cases, black arrows), while cases bearing the IGHV gene families 1-2, 1-3, 1-18, 1-8 associated with a very low constitutive VLA-4 activation state (21/134 cases, grey arrows). 4) Constitutive VLA-4 activation is hampered by IB exposure in-vivo - A decrease in constitutive VLA-4 activation was observed in CLL cells collected at pre-treatment and at day 14, 30 and 90 from patients undergoing IB treatment (Fig.F), suggesting an IB-dependent impairment of VLA-4 activation via BCR signal. 5) VLA-4 is activated via BCR autonomous signaling - Murine TKO cells express high level of cell surface VLA-4 (Fig.G). TKO cells expressing different BCRs derived from CLL cases (e.g. BCR17, a CLL- subset#2 case) show autonomous Ca++ influx as well as VLA-4 activation as compared to TKO cells expressing BCRs derived from healthy donor (BCR53, mature recirculating B cells). Notably, both BCR17 and BCR53, upon anti-IgM stimulation, induced high VLA-4 affinity state, while only BCR17 induced a significant increase of VLA-4 affinity in the absence of anti-IgM stimulation (Fig.H). Conclusion. The presence of a constitutively activated form of VLA-4 is observed in a significant fraction of CD49d+ CLL, due to a continuous VLA-4 inside-out stimulation derived from an autonomous BCR signaling. Figure Disclosures Di Raimondo: Takeda: Consultancy; Amgen: Consultancy, Honoraria, Research Funding. Rossi:Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Honoraria, Other: Scientific advisory board; Janseen: Honoraria, Other: Scientific advisory board; Roche: Honoraria, Other: Scientific advisory board; Astra Zeneca: Honoraria, Other: Scientific advisory board.

Association of Endothelial Biomarkers with Cerebral MRI Perfusion Analysis in Patients with Sickle Cell Disease

Introduction Sickle cell disease (SCD) is associated with silent cerebral infarcts (SCI) which are related to neurocognitive damage. One of the major causes of SCI is the impaired cerebral oxygenation, which makes cerebral blood flow (CBF) an essential parameter to measure. Previous hemodynamic studies have shown elevated CBF and reduced cerebrovascular reserve (CVR) in patients with SCD (Helton 2015; Václavů 2018). CVR is known as the increase of CBF in response to vasoactive stimulus, relative to the baseline. The reduced CVR renders SCD patients susceptible to cerebral ischemia, especially under hypotensive or hypoxic conditions. Possible factors contributing to microvascular damage and thus reduced CVR include hemoglobin S polymerization, neutrophil activation and endothelial activation and adhesion. Previous studies examined the role of these factors in patients with SCD (Sins 2017; Al Najjar 2017). However, the association between the endothelial biomarkers and hemodynamic parameters is unknown in adult patients with SCD. In this study, we investigated the correlation between CBF and CVR and the adhesion molecules including sVCAM-1, sP-selectin, VWF-Ag and ADAMTS13. Additionally, we studied the association of these endothelial biomarkers with standard laboratory parameters. Methods This study was performed in accordance with the Declaration of Helsinki and was approved by the Review Board of Amsterdam UMC. For this study, 33 steady state patients with SCD (mean age 32.1 ± 10.7, 64% male, 29 HbSS, 4 HbSß) and 10 healthy volunteers (mean age 36,4 ± 15.9, 60% male, 2 HbAS, 8 HbAA) were included. Hematologic laboratory parameters were assessed using standard laboratory procedures. Plasma levels of sVCAM-1 and sP-selectin were determined using ELISA (R&amp;D Systems, USA) and ADAMTS13 and VWF-Ag were measured with the INNOVANCE assay (Siemens Healthcare Diagnostics). For the CBF measurements, pseudo-continuous arterial spin labelling (pCASL) was acquired at 3T MRI (Philips Healthcare, The Netherlands). CBF was measured before and after acetazolamide (vasoactive stimulus) administration and subsequently CVR was calculated using the following equation: CVR = (CBFafter - CBFbefore) / CBFbefore x 100% Plasma levels of endothelial biomarkers were compared between groups using ANOVA test. Correlation between the parameters were measured using single regression model where p&lt;0.05 was considered as statistically significant. Results sVCAM-1 levels and VWF-Ag were significantly higher in SCD patients compared to healthy controls (p &lt; 0.01 and p = 0.01). ADAMTS13 and sP-selectin were not significantly different between the two groups (p = 0.06 and p = 0.33). sVCAM-1 was significantly associated with CBF, and parameters of hemolysis LDH and bilirubin in SCD patients (Fig. 1A and 2C). Negative correlation was observed between sVCAM-1 and hemoglobin (Fig. 1B). The relationship between sVCAM-1 and hemodynamic and laboratory parameters are shown in Table 1. No significant correlation was found between sVCAM-1, hemodynamic and standard laboratory parameters in healthy controls. VWF-Ag, ADAMTS13 and sP-selectin were not significantly associated with hemodynamic MRI parameters. Discussion Our results show elevated sVCAM-1 levels in sickle cell patients, strongly related to CBF. sVCAM-1 is an adhesion molecule and elevated plasma levels are found in patients with endothelial activation due to inflammation or atherosclerosis (Cook-Mills 2013; Cybulsky 2001). Previous studies showed elevated levels in sickle cell disease but no correlation with parameters of cerebral perfusion have been demonstrated yet (Antwi-Boasiako 2018; Kato 2005). The strong correlation with CBF is mostly related to the chronic hemolysis given the association found with hemoglobin levels and markers of hemolysis like LDH and bilirubin. In contrast to sVCAM-1, no such relation was found with other markers of endothelial activation such as VWF activity and sP-selectin levels. No correlation between endothelial markers and the CVR was demonstrated, suggesting that endothelial damage itself may not related to the impaired cerebral vasodilatation in response to a vasoactive stimulus. Conclusion Endothelial adhesion molecule sVCAM-1 showed a strong correlation with CBF and parameters of hemolysis, suggesting a relation between the hemolytic damage of endothelial cells and impaired cerebral perfusion. Disclosures Nur: Novartis Pharmaceuticals: Consultancy.

Adsorption of Soluble Immunoglobulin-Type Adhesion Molecules to Cellulose Acetate Beads.

Circulating levels of soluble intercellular adhesion molecule-1 (sICAM-1) and vascular adhesion molecule-1 (sVCAM-1) are elevated in patients with inflammatory bowel disease. Cellulose acetate (CA) beads are used as carriers for granulocyte and monocyte (GM) adsorptive apheresis (GMA). We investigated the effect of CA beads on sICAM-1 and sVCAM-1 plasma concentrations in vitro. Because GM adsorption to CA beads increased with a rise in the incubation temperature in our previous study, peripheral blood was incubated with and without CA beads at 5, 25, 37, or 43 °C and plasma sICAM-1 and sVCAM-1 was measured. The sICAM-1 and sVCAM-1 concentrations in samples incubated with CA beads were significantly lower than those without CA beads at all four temperatures. However, no significant differences were observed both sICAM-1 and sVCAM-1 plasma levels at the four different temperatures after incubation with CA beads. These results suggest that independent of incubation temperature, sICAM-1 and sVCAM-1 are likely to adsorb CA beads. These molecules may be a new index for predicting the therapeutic effects of GMA.

Soluble vascular cell adhesion molecular-1 is a potential biological indicator of hemophilic arthropathy.

Hemophilic arthropathy is the most common chronic complication in patients with hemophilia. The pathogenesis of hemophilic arthropathy involves the inflammatory processes associated with rheumatoid arthritis (RA). Determining the severity and/or progression of joint damage is crucial when evaluating the effect of treatment modalities. Identifying reliable biomarkers in the peripheral blood of patients with hemophilic arthropathy may be beneficial in clinical practice. Circulating soluble vascular cell adhesion molecule-1 (sVCAM-1), E-selectin, and P-selectin levels are elevated in patients with RA. Our study investigated whether these soluble adhesion molecules can be used as biological indicators in the course of joint damage in patients with hemophilia A.Patients with hemophilia A (mild, moderate, and severe) were enrolled. The plasma levels of sVCAM-1, E-selectin, and P-selectin in patients with hemophilia A and control were measured using specific enzyme-linked immunosorbent assay kits. Joint damages were evaluated using Pettersson scores.No statistically significant differences were observed in E-selectin and P-selectin levels between patients and controls. The sVCAM-1 level was significantly higher in patients with hemophilia A than in controls. The differences remained significant in patients with severe hemophilia A but not in patients with mild or moderate hemophilia A. The degree of hemophilic arthropathy was evaluated using Pettersson scores, and a score higher than 5 indicated marked arthropathy. Patients with more than 1 joint with marked arthropathy showed significantly higher sVCAM-1 levels.sVCAM-1 levels in patients with hemophilia A are associated with the severity of hemophilic arthropathy.

Soluble Vascular Cell Adhesion Molecule-1 (VCAM-1) as a Biomarker in the Mouse Model of Experimental Autoimmune Myocarditis (EAM).

Vascular cell adhesion molecule-1 (VCAM-1) is strongly upregulated in hearts of mice with coxsackie virus-induced as well as in patients with viral infection-triggered dilated cardiomyopathy. Nevertheless, the role of its soluble form as a biomarker in inflammatory heart diseases remains unclear. Therefore, we investigated whether plasma levels of soluble VCAM-1 (sVCAM-1) directly correlated with disease activity and progression of cardiac dysfunction in the mouse model of experimental autoimmune myocarditis (EAM). EAM was induced by immunization of BALB/c mice with heart-specific myosin-alpha heavy chain peptide together with complete Freund`s adjuvant. ELISA revealed strong expression of cardiac VCAM-1 (cVCAM-1) throughout the course of EAM in immunized mice compared to control animals. Furthermore, sVCAM-1 was elevated in the plasma of immunized compared to control mice at acute and chronic stages of the disease. sVCAM-1 did not correlate with the degree of acute cardiac inflammation analyzed by histology or cardiac cytokine expression investigated by ELISA. Nevertheless, heart to body weight ratio correlated significantly with sVCAM-1 at chronic stages of EAM. Cardiac systolic dysfunction studied with positron emission tomography indicated a weak relationship with sVCAM-1 at the chronic stage of the disease. Our data provide evidence that plasma levels of sVCAM-1 are elevated throughout all stages of the disease but showed no strong correlation with the severity of EAM.

P42. TNF-ALPHA gene polymorphism in pathology of preeclampsia

Introduction Preeclampsia (PE) is a life-threatening complication of pregnancy associated with a high rate of maternal and perinatal morbidity and/or mortality worldwide. If untreated, it can progress to eclampsia, which can result in the death of mother, fetus or both. Objectives Etiology of PE is still uncertain, however recently the role of immune system takes on importance. Thus the role of central cytokine, tumor necrosis factor (TNF) involved in inflammation processes become widely investigated in obstetric disorders. Following this the present study is to investigate the effect of TNF-alpha gene G308A (rs1800629) polymorphism on disease risk, renal functions, microvascular permeability, endothelial cell dysfunction and organ involvement in women with preeclampsia. Methods Initially a 102 3 rd trimester pregnant women (preeclamptic-cases and healthy controls) with singleton pregnancy were invited for participation of which 76 were genotyped for TNF-alpha G308A polymorphism, evaluated for plasma levels of sVCAM-1, fibronectin, TNF-alpha and correlated to profile of preeclampsia. The odds ratio (OR) and 95% confidence intervals obtained from unconditional logistic regression were used to set association between TNF-apolymorphism and PE risk. For continuous variables we applied Student's t -test and for categorical variables the Pearson x 2 or Fisher's exact test. The two-way ANOVA with Bonferroni correction was used in multivariate analysis. Results The A allele was more frequent in cases than controls (22.4% vs. 13.2%) what increased disease risk (OR=2.73). Maternal serum levels of TNF-alpha, sVCAM-1 and fibronectin were significantly increased in cases (855.8±385.1pg/mL, 1243±671ng/mL, 0.308±0.231g/L) compared to controls (301.1±156.1pg/mL, 651±250ng/mL, 0.218±0.101g/L, p p p =0.031; respectively) and levels shoved increasing trend with mutant allele in genotype. Moderate and severe proteinuria was higher in rs1800629 allele A subjects compared to G/G carriers (53.8% vs. 14.3%, p p Conclusion This study revealed possible association between clinical and laboratory manifestations of preeclampsia and TNF-alpha gene G308A (rs1800629) polymorphism.

Interference With TNFα Using Long-Term Etanercept In S+SAntilles Sickle Transgenic Mice Ameliorates Abnormal Endothelial Activation, Vasoocclusion, and Pulmonary Hypertension Including Its Pulmonary Arterial Wall Remodeling

A systemic inflammatory state is a prominent feature of sickle cell anemia. Using sickle transgenic mice, we previously identified a blood monocyte-TNF-endothelium activation axis using multiple complementary approaches. For example, endothelial activation (identified by abnormal expression of VCAM-1 and tissue factor (TF) by pulmonary vessel endothelium) was evident in unstressed BERK and S+SAntilles sickle mice. In NY1DD sickle mice, this was triggered by transient enhanced sickling and vasoocclusion resulting from transient exposure to hypoxia/reoxygenation (H/R). Endothelial activation depended upon Egr-1 and NFkB(p50) in peripheral blood mononuclear cells, and transfusion experiments clarified that the peripheral blood monocytes (PBM) were the responsible effecter cells. Finally, interference with TNFa using etanercept (Etn) prevented this PBM-induced endothelial activation. Based on these background data, we have now tested the “clinical” efficacy of long-term, anti-inflammatory TNFa interference via treatment of S+SAntilles sickle mice with Etn (started at weaning, 3 mg/kg sc, once/wk x 6 wk). In parallel, littermate controls received equimolar scrambled peptide version of Etn's TNF-receptor moiety. In response we observed significant improvement in multiple parameters relevant to inflammation-induced vessel wall disease in this model, as follows (data shown in inset). Etn decreased blood markers of inflammation: plasma levels of sVCAM-1 and SAP (by ELISA), and MCP-1 and TNFa (by FACS cytobead assay). Etn also decreased the % of PBM positive for TNFa (by FACS). Etn diminished the abnormal pulmonary vessel endothelial expression of VCAM-1 and TF (by histochemical and immunofluorescent staining). Etn eliminated the enhanced vasoocclusion seen as dermal microvascular stasis in the live mouse (using the dorsal skin fold chamber) measured at 1 and 4 hours post-H/R (7%O2x1hr, followed by 21%O2x 1-4hr). Instructively, Etn did not significantly improve the similar stasis induced by heme infusion, a TNF-independent direct endothelial stressor. Finally, Etn diminished evidence for pulmonary hypertension, assessed via three of its hallmark features: elevated right ventricular mean systolic pressure (by RV catheterization), abnormal peri-vascular inflammatory cell aggregates (by immuno-histochemical staining for CD11b), and abnormal pulmonary arterial wall muscularization (by immuno-histochemical staining for SM actin). Remarkably, Etn actually normalized RVSP, and Etn's partial improvement in pulmonary arterial muscularization resulted not only from prevention of its progression over duration of this study but also from some reversal of the abnormal remodeling that had already developed by the time Etn was started. In parallel, Etn reduced the number of abnormal peri-vascular aggregates of CDllb+ cells in the pulmonary arterial system.These results enable several conclusions. In the S+SAntilles sickle model, endothelial activation and aspects of vascular wall pathobiology derive from TNFa produced by activated PBM, a monocyte-TNF-endothelial activation axis. Interference with TNFa using etanercept exerts multiple salubrious vascular effects that predict possible clinical benefit. And results suggest that inflammation involving TNFa plays a proximate, generative role in development of pulmonary hypertension in this model. Assuming these benefits are independently corroborated, a controlled test of etanercept's efficacy for aspects of human sickle vascular pathobiology perhaps should be considered. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Effects of chocolate supplementation on metabolic and cardiovascular parameters in ApoE3L mice fed a high-cholesterol atherogenic diet

Dietary intake of cocoa and/or chocolate has been suggested to exhibit protective cardiovascular effects although this is still controversial. The aim of this study was to investigate the effects of chocolate supplementation on metabolic and cardiovascular parameters. Four groups of ApoE*3Leiden mice were exposed to the following diet regimens. Group 1: cholesterol-free control diet (CO). Group 2: high-dose (1.0% w/w) control cholesterol (CC). Group 3: CC supplemented chocolate A (CCA) and Group 4: CC supplemented chocolate B (CCB). Both chocolates differed in polyphenol and fiber content, CCA had a relatively high-polyphenol and low-fiber content compared to CCB. Mice fed a high-cholesterol diet showed increased plasma-cholesterol and developed atherosclerosis. Both chocolate treatments, particularly CCA, further increased plasma-cholesterol and increased atherosclerotic plaque formation. Moreover, compared to mice fed a high-cholesterol diet, both chocolate-treated groups displayed increased liver injury. Mice on high-cholesterol diet had elevated plasma levels of sVCAM-1, sE-selectin and SAA, which was further increased in the CCB group. Similar effects were observed for renal inflammation markers. The two chocolate preparations showed unfavorable, but different effects on cardiometabolic health in E3L mice, which dissimilarities may be related to differences in chocolate composition. We conclude that discrepancies reported on the effects of chocolate on cardiometabolic health may at least partly be due to differences in chocolate composition.

Abstract 72: Endothelial Cell Tissue Factor Enhances Inflammation but Not Coagulation in Mouse Model of Sickle Cell Disease

Sickle cell disease (SCD) is associated with increased tissue factor (TF) expression and activation of coagulation. Using a mouse model of SCD, we have previously demonstrated that inhibition of TF with a rat-anti mouse TF (1H1) antibody not only abolishes activation of coagulation (measured by plasma levels of thrombin anti-thrombin (TAT) complexes) but also reduces inflammation and endothelial cell (EC) injury, which was indicated by attenuation of plasma levels of IL-6 and sVCAM-1, respectively. SCD is one of the few diseases where TF has been shown to be expressed on ECs. Therefore, we investigated the effect of EC-specific deletion of TF gene on activation of coagulation, inflammation and EC injury in a mouse model of SCD (BERK mice). Sickle cell (SS) mice that expressed normal levels of TF or have EC-specific deletion of TF gene (TFfl/flEC) were generated by transplanting TFfl/fl (normal expression of TF) and TFfl/fl Tie2 Cre+ mice (lacking TF gene in both hematopoietic cells and ECs) with bone marrow from SS mice (bone marrow transplantation reconstituted expression of TF in all hematopoietic cells in TFfl/fl Tie2 Cre+). Interestingly, we found that activation of coagulation (TAT) and EC injury (sVCAM-1) were not affected by EC-specific deletion of TF gene. In contrast, plasma levels of IL-6 were significantly reduced in TFfl/flEC SS mice compared to SS mice with normal levels of TF expression (mean+/-SEM; 6.9+/-1.8 vs 14.9+/-3.0 pg/ml; p&lt;0.05). Similar results were observed in SS mice lacking protease activated receptor-2 (PAR-2) expression in all non-hematopoietic cells compared to SS mice expressing normal levels of PAR-2 (no difference in plasma levels of TAT and sVCAM-1; reduction of IL-6, 9.4+/-0.9 vs 18.9+/-4.5 pg/ml; p&lt;0.05). In contrast, SS mice with PAR-1 deficiency in all non-hematopoietic cells demonstrated reduced plasma levels of sVCAM-1 (835+/-41 vs 1003+/-52 ng/ml; p&lt;0.05) but not IL-6 or TAT. Our data indicates that increased expression of EC TF contributes to the inflammation (possibly via PAR-2 activation on ECs) whereas TF expressed by other cellular sources (monocytes,perivascular cells) contributes to the activation of coagulation and EC injury (likely via thrombin-dependent mechanisms) in this mouse model of SCD.

A potent oral P‐selectin blocking agent improves microcirculatory blood flow and a marker of endothelial cell injury in patients with sickle cell disease

Abnormal blood flow accounts for most of the clinical morbidity of sickle cell disease (SCD) [1,2]. Most notably, occlusion of flow in the microvasculature causes the acute pain crises [3] that are the commonest cause for patients with SCD to seek medical attention [4] and major determinants of their quality of life [5]. Based on evidence that endothelial P-selectin is central to the abnormal blood flow in SCD we provide results from four of our studies that are germane to microvascular blood flow in SCD. A proof-of-principle study established that doses of heparin lower than what are used for anticoagulation but sufficient to block P-selectin improved microvascular blood flow inpatients with SCD. An in vitro study showed that Pentosan Polysulfate Sodium (PPS) had greater P-selectin blocking activity than heparin. A Phase I clinical study demonstrated that a single oral dose of PPS increased microvascular blood flow in patients with SCD. A Phase II clinical study that was not completed documented that daily oral doses of PPS administered for 8 weeks lowered plasma levels of sVCAM-1 and tended to improve microvascular blood flow in patients with SCD. These data support the concept that P-selectin on the microvascular endothelium is critical to both acute vascular occlusion and chronically impaired microvascular blood flow in SCD. They also demonstrate that oral PPS is beneficial to microvascular sickle cell blood flow and has potential as an efficacious agent for long-term prophylactic therapy of SCD.

Plasma soluble vascular adhesion molecule-1 levels are persistently elevated during the first month after colorectal cancer resection

Plasma from the second and third weeks after minimally invasive colorectal resection (MICR) has high levels of the proangiogenic proteins VEGF and angiopoietin 2 and also stimulates, in vitro, endothelial cell (EC) proliferation and migration, which are critical to wound and tumor angiogenesis. Soluble vascular cell adhesion molecule-1 (sVCAM-1) stimulates EC chemotaxis and angiogenesis. The impact of MICR on blood levels of sVCAM-1 is unknown. This study's purpose was to determine plasma sVCAM-1 levels after MICR in colorectal cancer (CRC) patients. Blood samples from 90 patients (26% rectal, 74% colon) were obtained preoperatively, on postoperative days (POD) 1 and 3, and at other points during the next 2 months. The late samples were bundled into 7-day time blocks. sVCAM-1 levels were determined in duplicate via ELISA and reported as ng/ml. Student's t test was used for data analysis (significance, P < 0.008 after Bonferroni correction). The mean incision length was 7.3 ± 3.1 cm, and the conversion rate was 3%. Compared with preoperative (PreOp) levels (811.3 ± 233.2), the mean plasma sVCAM-1 level was significantly higher on POD 1 (905.7 ± 292.4, P < 0.001) and POD 3 (977.7 ± 271.8, P < 0.001). Levels remained significantly elevated for the POD 7-13, POD 14-20, POD 21-27, and POD 28-67 time blocks. MICR for CRC is associated with a persistent increase in plasma sVCAM-1 levels during the first month. This sustained increase may promote angiogenesis and stimulate the growth of residual tumor cells early after surgery.

Sunitinib‐induced changes in circulating endothelial cell‐related proteins in patients with metastatic renal cell cancer

Vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitors are effective agents in the treatment of metastatic renal cell cancer (mRCC). We here investigated whether inhibition of VEGFR signalin by sunitinib causes changes in plasma proteins associated with tumor endothelium. Forty-three patients with mRCC received sunitinib 50 mg/day in a 4-weeks on 2-weeks off schedule. Sequential plasma samples were obtained before treatment (C1D1), on C1D14, on C1D28, and on C2D1 before start of cycle 2. Plasma levels were assessed for VEGF, soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble intercellular cell adhesion molecule-1 (sICAM-1), von Willebrand factor (vWF), circulating angiopoietin-2 (Ang-2) and soluble Tie-2 (sTie-2). Total tumor burden was calculated at baseline and at first evaluation. Progression-free survival (PFS) and overall survival (OS) were determined. Tumor burden was positively associated with baseline circulating Ang-2 [Spearman's rho (ρ) = 0.378, p = 0.028] and vWF (ρ = 0.417, p = 0.008). During sunitinib treatment, circulating Ang-2 and sTie-2 significantly decreased (p < 0.001 for both), plasma levels of sVCAM-1 and VEGF significantly increased (p = 0.022 and p < 0.001), whereas those of sICAM-1 and vWF remained stable. These protein changes had recovered on C2D1. The reduction in circulating Ang-2 levels on C1D28 was positively correlated with the percentage decrease in tumor burden (ρ = 0.605; p = 0.002). Baseline protein levels and subsequent changes were not associated with PFS or OS. In conclusion, sunitinib-induced changes in Ang-2, sTie-2, sVCAM-1 and VEGF are related to the administration schedule, while reduction in Ang-2 is also associated with decrease in tumor burden.

Fetal endothelium dysfunction is associated with circulating maternal levels of sE-selectin, sVCAM1, and sFlt-1 during pre-eclampsia

Objectives. To evaluate the association between endothelial activation markers in the maternal circulation with nitric oxide (NO) synthesis in human umbilical endothelial cells.Study design. This is a case-control study of normal and pre-eclamptic pregnancies. The levels of sE-selectin, soluble vascular cell adhesion molecule 1 (sVCAM-1), and soluble fms-like tyrosine kinase 1 (sFlt-1) were measured by enzyme-linked immunosorbent assay, and histamine-induced NO synthesis was detected by fluorometric examination of the human umbilical vein endothelial cells (HUVECs) isolated from normal and pathological pregnancies.Results. Mothers with severe pre-eclamptic pregnancies have premature and smaller babies than mothers with normal pregnancies (P < 0.05); they also have high maternal plasma levels of sVCAM-1 (∼2-fold), sFlt-1 (∼2.5-fold), and lower (∼70%) histamine-stimulated NO synthesis in HUVECs. A positive relationship between systolic blood pressure (SBP) and plasma levels of sE-selectin, sVCAM-1, and sFlt-1 was demonstrated. Moreover, levels of sE-selectin, sVCAM-1, and sFlt-1 were negatively associated with newborn weight (NBW), gestational age at delivery, and NO synthesis. Women with high E-selectin (>63 ng/ml), VCAM-1 (>752 ng/ml), and sFlt-1 (>15204 pg/ml) showed high risk (∼2-fold) for preterm delivery and very preterm delivery, or fetal weight <1500 g (∼1.5-fold) compared with women with low levels.Conclusions. High circulating levels of maternal endothelial dysfunction markers present in pre-eclampsia are associated with decreased NO synthesis in fetal endothelium.

Increased Plasma and Endothelial Cell Expression of Chemokines and Adhesion Molecules in Chronic Kidney Disease

Chemokines and adhesion molecules are involved in early events of atherogenesis. In the present study, we investigated the effects of the uremic milieu on the expression of monocyte chemoattractant protein-1 (MCP-1), interleukin-8 (IL-8), soluble vascular adhesion molecule-1 (sVCAM-1) and soluble intercellular adhesion molecule-1 (sICAM-1) and their relationship to cardiovascular status. Plasma samples were obtained from patients in different stages of chronic kidney disease (CKD). Cardiovascular status was evaluated by intima-media thickness and endothelial dysfunction by flow mediation dilatation and proteinuria. In vitro studies were performed using human umbilical endothelial cells exposed to uremic plasma or plasma from healthy subjects. MCP-1, IL-8, sVCAM-1 and sICAM-1 levels in plasma and in supernatant were analyzed by enzyme-linked immunosorbent assay. The population consisted of 73 (mean age 57 years; 48% males) CKD patients with glomerular filtration rate (GFR) of 37 ± 2 ml/min. MCP-1 and sVCAM-1 plasma levels were negatively correlated with GFR (ρ = –0.40, p < 0.0005 and ρ = –0.42, p < 0.0005, respectively). Fibrinogen was positively correlated with MCP-1, sICAM-1 and sVCAM-1 (ρ = 0.33, p < 0.005, ρ = 0.32, p < 0.05 and ρ = 0.25, p < 0.05, respectively) and ultra-high-sensitivity C-reactive protein was positively correlated with sICAM-1 (ρ = 0.25, p < 0.0005). Plasma IL-8 had a significant positive correlation with proteinuria (ρ = 0.31, p < 0.01). There was a time- and CKD-stage-dependent MCP-1, IL-8 and sVCAM-1 endothelial expression (p < 0.05). In summary, plasma levels of markers of endothelial cell activation (MCP-1 and sVCAM-1) are increased in more advanced CKD. Exposure of endothelial cells to uremic plasma results in a time- and CKD-stage-dependent increased expression of MCP-1, IL-8 and sVCAM-1, suggesting a link between vascular activation, systemic inflammation and uremic toxicity. Future studies are necessary to investigate whether these biomarkers add predictive value in comparison to the previously described ones. Also, endothelial response to uremic toxicity should be viewed as a potential target for intervention in order to reduce morbidity and mortality in CKD-related cardiovascular disease.