Fariborz Mobarrez

Atorvastatin reduces thrombin generation and expression of tissue factor, P-selectin and GPIIIa on platelet-derived microparticles in patients with peripheral arterial occlusive disease

We investigated the effects of statin treatment on platelet-derived microparticles (PMPs) and thrombin generation in atherothrombotic disease. Nineteen patients with peripheral arterial occlusive disease were randomised to eight weeks of treatment with atorvastatin or placebo in a cross-over fashion. Expression of GPIIIa (CD61), P-selectin (CD62P), tissue factor (TF, CD142) and phosphatidylserine (PS; annexin-V or lactadherin binding) was assessed on PMPs. Thrombin generation in vivo was assessed by measurement of prothrombin fragment 1+2 in plasma (F1+2) and ex vivo by using the calibrated automated thrombogram (CAT). During atorvastatin treatment, expression of TF, P-selectin and GPIIIa was significantly reduced vs. placebo (p<0.001 for all). No effect on annexin-V or lactadherin binding was seen. Thrombin generation was significantly reduced during atorvastatin as assessed by both the CAT assay (p<0.001) and by measurements of F1+2 (p<0.01). Subsequent in vitro experiments showed that when TF on microparticles (MPs) was blocked by antibodies, the initiation of thrombin generation was slightly but significantly delayed. Blocking PS on MPs using annexin-V or lactadherin resulted in almost complete inhibition of thrombin generation. In conclusion, atorvastatin reduces thrombin generation and expression of TF, GPIIIa and P-selectin on PMPs in patients with peripheral vascular disease. Microparticle-bound TF slightly enhances initiation of thrombin generation whereas negatively charged surfaces provided by MPs or lipoproteins could reinforce thrombin generation. Statins may inhibit initiation of thrombin generation partly through a microparticle dependent mechanism but the main effect is probably through reduction of lipoprotein levels.

Low plasma vascular endothelial growth factor (VEGF) associated with completed suicide

Abstract Objectives. Immunological differences have previously been associated with depression and suicidal behaviour. Several cytokines have been identified as potentially important in understanding the pathophysiology of mood disorders and suicidality. Here we aimed to identify new inflammatory biomarkers for suicide prediction. Methods. Plasma concentrations of interleukin (IL) 1-a , IL1-b, IL-2, IL-4, IL-6, IL-8, IL-10, interferon-gamma (IFNG), tumor necrosis factor-a (TNF-a), monocyte chemotactic protein-1 (MCP-1), epidermal growth factor (EGF), and vascular endothelial growth factor (VEGF) were measured in 58 suicide attempters with a high throughput automated biochip immunoassay system. Patients were evaluated using the Montgomery–Åsberg Depression Rating Scale (MADRS) and the Suicide Intent Scale (SIS). All patients were followed up for cause of death. Results. We found significantly lower levels of VEGF in the seven patients who upon a mean follow-up of 13 years were found to have completed suicide. VEGF also showed a trend for negative correlation with the planning subscale of SIS. A trend could be shown for lower IL-2 and for higher IFNG levels in suicide victims. Conclusions. Our study provides further support for a role of inflammation in the pathophysiology of suicidality. VEGF may be related with suicide risk.

Atorvastatin has antithrombotic effects in patients with type 1 diabetes and dyslipidemia.

INTRODUCTION Diabetes is a prothrombotic state involving a more thrombogenic fibrin network. In the present study we investigated the effects of lipid-lowering therapy with atorvastatin on fibrin network structure and platelet-derived microparticles in patients with type 1 diabetes and dyslipidemia. MATERIALS AND METHODS Twenty patients were treated with atorvastatin (80 mg daily) or placebo during 2 months in a randomized, double-blind, cross-over study. Fibrin network permeability, expression of glycoprotein IIIa, P-selectin and tissue factor on platelet-derived microparticles, plasma endogenous thrombin potential, plasminogen activator inhibitor-1 and tissue plasminogen activator antigen levels were assessed. Additionally, levels of plasma fibrinogen, high-sensitivity C-reactive protein and glycated haemoglobin were measured. RESULTS During treatment with atorvastatin, fibrin network permeability increased (p=0.01), while endogenous thrombin potential and expression of glycoprotein IIIa, P-selectin and tissue factor decreased (p<0.01). In vitro experiments indicated that platelet-derived microparticles influence the fibrin network formation as fibrin network permeability decreased significantly when platelet-derived microparticles were added to normal plasma. Baseline levels of plasminogen activator inhibitor-1 and tissue plasminogen activator antigen as well as plasma fibrinogen and high-sensitivity C-reactive protein were within reference values and not significantly changed during atorvastatin treatment, while glycated haemoglobin increased 0.3% (p<0.001). CONCLUSIONS Novel treatment effects were found in patients with type 1 diabetes and dyslipidemia during atorvastatin therapy, i.e. a more porous fibrin network, to which reduced expression of glycoprotein IIIa, P-selectin and tissue factor on platelet-derived microparticles may contribute. The observed impairment of glycemic control during long-term statin treatment deserves attention.

Platelet-derived microparticles during and after acute coronary syndrome.

As microparticles are shedded upon platelet activation, and may be used to assess platelet function, we measured plasma concentrations of platelet-derived microparticles (PMPs) during and after an acute coronary syndrome (ACS). Fifty-one patients with ACS were investigated at admission, within 24 hours (before coronary angiography), and six months later. Sixty-one sex- and age-matched healthy controls were investigated once. PMPs were defined as particles <1.0 μm in size, negative to phalloidin (labels cell-fragments), and positive to CD61. Exposure of phosphatidylserine (PS+), CD62P and CD142 were also measured. Plasma concentrations of PS+PMPs exposing CD61, CD62P and CD142 were elevated 2.5, 6.0-, and 5.0-fold at admission (p<0.001 for all, compared to controls; aspirin only), decreased significantly 24 hours later following initiation of treatment with clopidogrel and subcutaneous anticoagulation (p<0.001 for all), and decreased even further six months later (p<0.01 for all). However, PS+PMPs exposing CD62P or CD142 were still between 1.2-and 2.3-fold higher than in controls (p<0.001 for both). The pattern for PS-PMPs during and after the ACS was very similar to that for PS+PMPs although the numbers were approximately 1/3 lower. In conclusion, PMP concentrations follow the pattern of platelet activation during and after an ACS. Decreased concentrations are observed after initiation of antithrombotic treatment, but PMP exposing CD62P or CD142 are still elevated after six months. Flow cytometric measurements of PMP in frozen-thawed samples enable studies of platelet function in larger clinical trials.

Low vascular endothelial growth factor and interleukin-8 in cerebrospinal fluid of suicide attempters.

A dysregulated immune system influencing pathways for cytokine regulation and growth factor expression is implicated in the pathophysiology of several neuropsychiatric disorders. Here, we analyzed cerebrospinal fluid (CSF) cytokines and growth factors with an ultra-sensitive immunoassay system in 43 medication-free suicide attempters and 20 healthy male volunteers. CSF vascular endothelial growth factor (VEGF) and CSF interleukin-8 (IL-8) levels were significantly lower in suicide attempters compared with healthy controls. Further, CSF VEGF showed a significant negative correlation with depression severity. CSF IL-6 levels did not differ between suicide attempters and healthy controls. Low CSF levels of VEGF may represent a lack of trophic support to neurons and downregulation of neurogenesis in the hippocampus reflecting more severe depressive states. IL-8 has also been reported as important in neuroprotection as well as having chemokine activity in the innate immune response. The results support a role for an impaired innate immunity and dysregulation of neuroprotection in the pathophysiology of depression and suicidal behavior.

The Effects of Smoking on Levels of Endothelial Progenitor Cells and Microparticles in the Blood of Healthy Volunteers

Background Cigarette smoking, both active and passive, is one of the leading causes of morbidity and mortality in cardiovascular disease. To assess the impact of brief smoking on the vasculature, we determined levels of circulating endothelial progenitor cells (EPCs) and circulating microparticles (MPs) following the smoking of one cigarette by young, healthy intermittent smokers. Materials and Methods 12 healthy volunteers were randomized to either smoking or not smoking in a crossover fashion. Blood sampling was performed at baseline, 1, 4 and 24 hours following smoking/not smoking. The numbers of EPCs and MPs were determined by flow cytometry. MPs were measured from platelets, leukocytes and endothelial cells. Moreover, MPs were also labelled with anti-HMGB1 and SYTO 13 to assess the content of nuclear molecules. Results Active smoking of one cigarette caused an immediate and significant increase in the numbers of circulating EPCs and MPs of platelet-, endothelial- and leukocyte origin. Levels of MPs containing nuclear molecules were increased, of which the majority were positive for CD41 and CD45 (platelet- and leukocyte origin). CD144 (VE-cadherin) or HMGB1 release did not significantly change during active smoking. Conclusion Brief active smoking of one cigarette generated an acute release of EPC and MPs, of which the latter contained nuclear matter. Together, these results demonstrate acute effects of cigarette smoke on endothelial, platelet and leukocyte function as well as injury to the vascular wall.

Pro-inflammatory cytokines are elevated in adolescent females with emotional disorders not treated with SSRIs

BACKGROUND Adults with major depressive disorder (MDD) show elevated levels of IL-6 and TNF-alpha. Studies of adolescents with MDD or anxiety disorders (AD) are few and present conflicting results. METHODS We studied plasma cytokines in a clinical sample of adolescent females with MDD and/or clinical AD (n=60, mean age 16.8 years), compared to healthy controls (n=44; mean age 16.5 years). RESULTS The clinical sample showed significantly higher values of IL-2 (Z=-4.09, p>0.0001), IL1-beta (Z=-2.40, p<0.05) and IL-10 (Z=-2.38, p<0.05) as compared to controls. The subgroup of the clinical sample not treated with SSRIs had a significant difference of IL-6 (Z=-2.26, p<0.05) in addition to the difference of IL-2 and IL1-beta, but showed no difference of IL-10 as compared to the controls. SSRI treatment was related to IL-6, explaining 26% of the variance in the clinical sample after controlling for BMI and symptom severity. In the clinical sample, levels of IL-6 and IFN-gamma were positively correlated with self-assessed symptoms of anxiety and/or depression (corr.coeff 0.35 resp 0.40 at p<0.05). LIMITATIONS The cross-sectional design does not allow for conclusions on causality. The sample sizes were relatively small and a large drop-out in the clinical sample may have influenced the representativity. DISCUSSION The study suggests that pro-inflammatory cytokines are part of the pathophysiology of emotional disorders in adolescent females and that SSRIs have anti-inflammatory properties. The findings prompt further studies on the specific mechanisms involved and may contribute to the development of more effective treatment and prevention.

Formation of Microparticles in the Injured Brain of Patients with Severe Isolated Traumatic Brain Injury

The potential pathophysiological role of circulating microparticles (MPs) has been recognized in various conditions, such as cardiovascular and thrombotic diseases. Traumatic brain injury (TBI) has a complex pathophysiology that involves coagulopathy and inflammation. We investigated endothelial-, platelet-, and leukocyte-derived microparticles (EMPs, PMPs, and LMPs, respectively) in 16 patients with severe isolated TBI. Arterial and cerebrovenous samples were taken repeatedly, during 1-72 h after injury. Subpopulations of MPs, exposing tissue factor (TF) and P-selection, were also studied. MP counts in cerebrovenous samples, irrespective of cellular origin, were higher in TBI cases, compared to healthy controls (peak levels of EMPs were approximately 7 times higher, PMPs 1.4 times higher, and LMPs 2 times higher, respectively; p<0.001 for all). MP counts declined sharply from high levels shortly after the trauma toward slightly elevated levels 72 h later. EMPs and PMPs exposing TF, as well as PMPs exposing P-selection, showed a transcranial gradient with higher concentration in cerebrovenous, compared to arterial, samples. In contrast, LMPs exposing TF were higher in arterial samples, suggesting accumulation of LMPs in the brain. We conclude that the pattern of circulating MPs is altered after TBI. PMPs exposing P-selection and EMPs exposing TF seem to be generated in the injured brain, whereas LMPs exposing TF are accumulated. The pathophysiological significance of these changes in MP pattern in TBI should be further investigated. Including MPs exposing brain-specific antigens in the assessment of brain injury would give further information of origin and likely give additional information of the size of the injury, given that the MP phenotypes investigated in the present study are not brain-specific markers.

Effect of lipopolysaccharide administration on the number, phenotype and content of nuclear molecules in blood microparticles of normal human subjects.

Microparticles (MPs) are small membrane‐bound vesicles that arise from activated and dying cells and promote inflammation and thrombosis. To characterize the in vivo release of MPs, we used flow cytometry to measure MPs in the blood of 15 healthy volunteers administered bacterial endotoxin (lipopolysaccharide or LPS) in the presence of a low dose of hydrocortisone with or without inhaled nitric oxide. MPs, defined as particles less than 1.0 μm in size, were assessed following labelling for CD42a, CD14 and CD62E or CD144 antibodies to identify MPs from platelets (PMPs), monocytes (MMPs) and endothelial cells (EMPs). In addition, PMPs and MMPs were labelled with anti‐HMGB1 and stained with SYTO13 to assess nuclear acid content. Administration of LPS led to an increase in the numbers of PMPs, MMPs and EMPs as defined by CD62E, as well as the number of MMPs and PMPs staining with anti‐HMGB1 and SYTO13. Inhalation of NO did not influence these findings. Together, these studies show that LPS can increase levels of blood MPs and influence phenotype, including nuclear content. As such, particles may be a source of HMGB1 and other nuclear molecules in the blood during inflammation.

Studies of microparticles in patients with the antiphospholipid syndrome (APS)

Objectives: To study circulating platelet, monocyte and endothelial microparticles (PMPs, MMPs and EMPs) in patients with antiphospholipid syndrome (APS) in comparison with healthy controls. Material and method: Fifty-two patients with APS and 52 healthy controls were investigated. MPs were measured on a flow cytometer (Beckman Gallios) and defined as particles sized < 1.0 µm, negative to phalloidin, positive to lactadherin and positive to either CD42a (PMPs), CD144 (EMPs) or CD14 (MMPs). Exposure of CD142 (TF) was measured on CD144 positive MPs. Results: Total number of MPs (i.e. lactadherin positive particles) was higher in APS patients versus controls (p < 0.001). An increased number of EMPs (p < 0.001), increased TF-positive EMPs (p < 0.001) and increased MMPs (p < 0.001) were also observed. PMP numbers did not differ between the groups. None of the MP types differed in numbers between obstetric and thrombotic APS patients. Conclusion: We observed a high number of EMPs expressing TF in APS patients. The numbers of MMPs and total EMPs were also higher as compared with healthy controls but in contrast to previous reports, the number of PMPs did not differ between groups.