Angiogenic factors in atrial fibrillation: a possible role in thrombogenesis?

BACKGROUND: The precise pathophysiological processes underlying the prothrombotic or hypercoagulable state in atrial fibrillation (AF) remain uncertain. We hypothesized a relationship between abnormal endothelial damage/dysfunction, coagulation, and angiogenic factors, thereby contributing to increased thrombogenicity. METHODS: Plasma levels of von Willebrand factor (vWF, an index of endothelial damage/dysfunction) and tissue factor (TF, an index of coagulation), as well as the angiogenic factors, vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2), were measured by enzyme-linked immunosorbant assay (ELISA) in 59 chronic AF patients. Data were compared to 40 age- and sex-matched healthy controls in sinus rhythm. RESULTS: Plasma vWF, VEGF and Ang-2 were significantly higher in AF patients compared to healthy controls (P=0.005, P=0.0055 and P<0.0001 respectively) but there were no significant differences in plasma Ang-1 or TF levels between the two groups (P=0.925 and P=0.121 respectively). Significant correlations were found between VEGF and vWF levels (Spearman, r=0.262, P=0.011) and between VEGF and Ang-2 (r=0.333, P=0.001). CONCLUSIONS: Raised VEGF in association with Ang-2 and vWF may reflect a link between abnormal endothelial damage/dysfunction and angiogenic factors. These may act together to alter TF expression and endothelial integrity, thereby contributing to the prothrombotic state in AF.     

Platelet‐derived VEGF,Flt‐1, Angiopoietin‐1 and P‐selectin in breast and prostate cancer: further evidence for a role of platelets in tumour angiogenesis

BACKGROUND: Cancer is a complex multi‐factorial disorder that may commonly show abnormal angiogenesis in such patients. Recently, platelets have been postulated to have a major role in both these processes, suggesting that anti‐platelet strategies may be useful in cancer treatment.

MATERIALS AND METHODS: To further investigate the role of platelets in angiogenesis, we used a novel platelet lysate assay to analyse platelet contents in breast cancer (n?=?30) and prostate cancer (n?=?30) patients and age‐ and sex‐matched controls (n?=?60). Markers of angiogenesis (vascular endothelial growth factor (VEGF), angiopoietin‐1 and–2 (Ang‐1, ‐2), and their respective receptors (Flt‐1 and Tie‐2) plus a marker of platelet activation (P‐selectin (P‐sel)), were all measured in platelet lysate by enzyme‐linked immunsorbent assay.

RESULTS: Platelet lysate from breast cancer patients contained higher levels of VEGF (P?<?0.0001), Ang‐1 (P?=?0.0186) and P‐sel (P?=?0.0002), compared to healthy controls. Platelet lysate from prostate cancer patients had elevated VEGF (P?=?0.008) but not Ang‐1 or P‐sel. There were no significant differences between levels of Flt‐1 between patients and controls, and both Ang‐2 and Tie‐2 were undetectable in both patient groups and control platelet lysate.

CONCLUSION: We have shown that our previously developed platelet lysate technique could be used to measure indices of angiogenesis, and their respective receptors, and that this assay can be applied to patients with cancer. Our study also provides further evidence that platelets may influence angiogenic abnormalities in human cancer. The platelet may be a useful target in anti‐cancer strategies.     

The effects of exercise stress testing on soluble E‐selectin,von Willebrand factor,and circulating endothelial cells as indices of endothelial damage/dysfunction

Background. The quantification of circulating endothelial cells (CECs) in whole blood is an increasingly recognized index of endothelial damage/dysfunction. Abnormal CECs have been linked to the severity of coronary artery disease (CAD).

Objective. We assessed the relationship of CECs to other markers of endothelial dysfunction (von Willebrand factor (vWF) and soluble E‐selectin (sEsel)) during exercise stress testing (EST) in a cohort of patients with suspected CAD.

Methods. We studied a cohort of patients referred to our chest pain clinic with a history of exertional chest pain. Treadmill EST was performed, using a full Bruce exercise protocol. Blood for CECs (immunobead method), vWF and sEsel (both ELISA) were collected immediately before (pre‐exercise), immediately following exercise, and at 30 minutes post‐EST.

Results. We studied 31 patients (84% male; mean (SD) age 58.4 (9.8) years). Of the entire cohort, 14 patients (45.2%) had a positive EST. Exercise led to significant increases in levels of CECs, sEsel, vWF, white blood cells (WBC), heart rate, mean and systolic blood pressure compared with base‐line (all P<0.05). There was a significant correlation between the change (Δ (immediate post–pre‐exercise)) in CECs and ΔvWF (r = 0.45; 95% CI 0.11–0.69: P = 0.01) and ΔsEsel (r = 0.41; 0.05–0.7: P = 0.02), as well as between ΔvWF and ΔsEsel (r = 0.55; 0.25–0.76: P = 0.001). Neither absolute nor ΔCEC counts were predictive of exercise work‐load/functional capacity, nor the presence of positive EST results.

Conclusion. EST led to a significant increase in endothelial markers (CECs, vWF, and sEsel) compared with base‐line levels. The rise in CECs correlated with the increases in other endothelial markers, but was not related to the either exercise work‐load/capacity or to the presence of a positive EST.     

Indices of angiogenesis,platelet activation,and endothelial damage/dysfunction in relation to ethnicity and coronary artery disease: Differences in central versus peripheral levels

Background. We hypothesized that indices of angiogenesis (vascular endothelial growth factor (VEGF), angiopoietins (Ang‐1 and ‐2), platelet activation (soluble P‐selectin)) and endothelial damage/dysfunction (von Willebrand factor (vWf)) would be more deranged in South Asians than in white Europeans when measured within the coronary sinus or coronary artery per se (that is, intracardiac sampling of blood supplying and draining the heart), as compared to measurements from the peripheral venous system.

Methods. To test this hypothesis, we performed a cross‐sectional study of 87 subjects undergoing cardiac catheterization, where 43 were South Asian and 44 were white European.

Results. South Asian participants were younger (P = 0.01) but had a lower rate of self‐reported smoking (P = 0.01). The extent of coronary atherosclerosis, assessed using presence of lesions>50%, number of vessels diseased and Gensini score, was comparable between the two ethnic groups (all P = NS). When samples were analysed from the coronary circulation or the femoral vein in relation to South Asian and white European ethnicity, there were no significant differences in the levels of VEGF, angiopoietins 1 and 2, soluble P‐selectin and vWf levels between the two ethnic groups.

Conclusion. Indices of angiogenesis, platelet activation, and endothelial damage/dysfunction are comparable in South Asians and their white European counterparts. Our results suggest that their pathophysiological roles may be comparable in South Asians and white Europeans in the context of coronary artery disease.     

Defining conditions for covalent immobilization of angiogenic growth factors onto scaffolds for tissue engineering

Rapid vascularization of engineered tissues in vitro and in vivo remains one of the key limitations in tissue engineering. We propose that angiogenic growth factors covalently immobilized on scaffolds for tissue engineering can be used to accomplish this goal. The main objectives of this work were: (a) to derive desirable experimental conditions for the covalent immobilization of vascular endothelial growth factor (VEGF) and angiopoietin‐1 (Ang1) on porous collagen scaffolds; and (b) to determine whether primary endothelial cells respond to these scaffolds with covalently immobilized angiogenic factors. VEGF and Ang1 were covalently immobilized onto porous collagen scaffolds, using 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide hydrochloride (EDC) chemistry. To improve covalent immobilization conditions: (a) different reaction buffers [phosphate‐buffered saline (PBS), distilled water, or 2‐(N‐morpholino)ethanesulphonic acid (MES)] were used; and (b) step immobilization was compared to bulk immobilization. In step immobilization, growth factors are applied after EDC activation of the scaffold, while in bulk immobilization, reagents are simultaneously applied to the scaffold. PBS as the reaction buffer resulted in higher amounts of VEGF and Ang1 immobilized (ELISA), higher cell proliferation rates (XTT) and increased lactate metabolism compared to water and MES as the reaction buffers. Step immobilization in PBS buffer was also more effective than bulk immobilization. Immobilized growth factors resulted in higher cell proliferation and lactate metabolism compared to soluble growth factors used at comparable concentrations. Tube formation by CD31‐positive cells was also observed in collagen scaffolds with immobilized VEGF or Ang1 using H5V and primary rat aortic endothelial cells but not on control scaffolds. Copyright © 2010 John Wiley & Sons, Ltd.     

Systemic and intracardiac vascular endothelial growth factor and angiopoietin‐1 and ‐2 levels in coronary artery disease: Effects of angioplasty

Background. Vascular growth factors are involved in the pathophysiology of human atherosclerotic vascular disease and plaque destabilization. We hypothesized that in stable patients with coronary artery disease (CAD), plasma levels of vascular endothelial growth factor (VEGF) and angiopoietins 1 and 2 (as indices of angiogenesis) would be no higher in coronary sinus blood when compared to the aortic root, coronary ostium, and peripheral femoral vein. Secondly, we hypothesized that percutaneous coronary intervention (PCI; angioplasty±stenting) would increase intracardiac levels of these indices, perhaps by destabilizing coronary plaques.

Methods. Patients undergoing elective diagnostic coronary angiography (n = 70; mean age 58.8±11.2 years) of which 37 proceeded to PCI were recruited. Blood samples were obtained from the aortic root, coronary ostium, coronary sinus, and femoral vein. Plasma VEGF, angiopoietin‐1 and angiopoietin‐2 levels were measured by immunoassays.

Results. There were no significant differences in VEGF, angiopoietin‐1 and angiopoietin‐2 levels when aortic root, coronary ostium, coronary sinus, and femoral vein samples were compared (P = not significant (NS)). In patients undergoing PCI, peripheral angiopoietin‐2 levels were increased significantly post PCI (P = 0.01). There was also a difference in intracardiac gradient (that is, aortic root‐coronary sinus difference) in angiopoietin‐1 (P = 0.02) following PCI. No significant changes in VEGF with PCI were noted.

Conclusion. There were no differences in indices of angiogenesis when aortic root, coronary ostium, coronary sinus, and femoral vein levels of VEGF and angiopoietins are compared, suggesting that peripheral blood measurements of these indices are comparable to intracardiac levels. Although no immediate effects were observed in soluble VEGF levels, PCI affected intracardiac angiopoietin‐1 with a systemic release of angiopoietin‐2. Further investigations are necessary to determine the relative systemic and intracardiac effects of the angiopoietins in vascular remodelling post PCI.     

Conditioned media from hypoxic‐cultured human dental pulp cells promotes bone healing during distraction osteogenesis

Distraction osteogenesis (DO) is a surgical procedure used to correct various skeletal disorders. Improving the technique by reducing the healing time would be of clinical relevance. The aim of this study was to determine the angiogenic and regenerative potential of conditioned media (CMs) collected from human dental pulp cells (hDPCs) grown under different culture conditions. CM collected from cells under hypoxia was used to improve bone healing and the DO procedure in vivo . The angiogenic potentials of CMs collected from hDPCs grown under normoxic (?Nor) and hypoxic (?Hyp) conditions were evaluated by quantitative PCR (VEGF‐A , angiopoietin‐1, angiopoietin‐2, interleukin‐6 (IL‐6 ) and CXCL12 ), ELISA assays (VEGF‐A, Ang‐2), tube‐formation and wound‐healing assays, using human umbilical vein endothelial cells. The results demonstrated that hypoxic CM had significantly higher angiogenic potential than normoxic CM. Human fetal osteoblasts (hFOBs) were exposed to CM, followed by alizarin red staining, to assess the osteogenic potential. It was found that CM did not enhance the mineralization capacity of hFOBs. DO was performed in the tibiae of 30 mice, followed by a local injection of 20 µl CM (CM–Nor and CM–Hyp groups) or serum‐free DMEM (control group) into the distraction zone every second day. The mice were sacrificed at days 13 and 27. The CM–Hyp treatment revealed a higher X‐ray density than the control group (p < 0.05). Our study suggests that the angiogenic effect promoted by hypoxic culture conditions is dependent on VEGF‐A and Ang‐2 released from hDPCs. Furthermore, CM–Hyp treatment may thus improve the DO procedure, accelerating bone healing. © 2015 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons, Ltd.     

Angiogenic factors and their soluble receptors predict organ dysfunction and mortality in post-cardiac arrest syndrome

Introduction

Post-cardiac arrest syndrome (PCAS) often leads to multiple organ dysfunction syndrome (MODS) with a poor prognosis. Endothelial and leukocyte activation after whole-body ischemia/reperfusion following resuscitation from cardiac arrest is a critical step in endothelial injury and related organ damage. Angiogenic factors, including vascular endothelial growth factor (VEGF) and angiopoietin (Ang), and their receptors play crucial roles in endothelial growth, survival signals, pathological angiogenesis and microvascular permeability. The aim of this study was to confirm the efficacy of angiogenic factors and their soluble receptors in predicting organ dysfunction and mortality in patients with PCAS.

Methods

A total of 52 resuscitated patients were divided into two subgroups: 23 survivors and 29 non-survivors. The serum levels of VEGF, soluble VEGF receptor (sVEGFR)1, sVEGFR2, Ang1, Ang2 and soluble Tie2 (sTie2) were measured at the time of admission (Day 1) and on Day 3 and Day 5. The ratio of Ang2 to Ang1 (Ang2/Ang1) was also calculated. This study compared the levels of angiogenic factors and their soluble receptors between survivors and non-survivors, and evaluated the predictive value of these factors for organ dysfunction and 28-day mortality.

Results

The non-survivors demonstrated more severe degrees of organ dysfunction and a higher prevalence of MODS. Non-survivors showed significant increases in the Ang2 levels and the Ang2/Ang1 ratios compared to survivors. A stepwise logistic regression analysis demonstrated that the Ang2 levels or the Ang2/Ang1 ratios on Day 1 independently predicted the 28-day mortality. The receiver operating characteristic curves of the Ang2 levels, and the Ang2/Ang1 ratios on Day 1 were good predictors of 28-day mortality. The Ang2 levels also independently predicted increases in the Sequential Organ Failure Assessment (SOFA) scores.

Conclusions

We observed a marked imbalance between Ang1 and Ang2 in favor of Ang2 in PCAS patients, and the effect was more prominent in non-survivors. Angiogenic factors and their soluble receptors, particularly Ang2 and Ang2/Ang1, are considered to be valuable predictive biomarkers in the development of organ dysfunction and poor outcomes in PCAS patients.     

Plasma vascular endothelial growth factor, angiopoietin-1, and angiopoietin-2 in diabetes: implications for cardiovascular risk and effects of multifactorial intervention

OBJECTIVE: Vascular endothelial growth factor (VEGF) and angiopoietin (Ang)-1 and Ang-2 are mediators of angiogenesis. More recent data suggest that the balance between these growth factors may affect vascular endothelial integrity. Because diabetes is closely associated with endothelial perturbation, we studied plasma levels of these angiogenic growth factors in patients with diabetes; their relationship with glycemia, inflammation, and endothelial damage/dysfunction; and the effect of intensified cardiovascular risk management. RESEARCH DESIGN AND METHODS: We measured plasma VEGF, Ang-1, and Ang-2 alongside plasma von Willebrand factor (vWf) and urine albumin-to-creatinine ratio (marking endothelial damage/dysfunction) and interleukin (IL)-6 in 94 patients (38 with overt cardiovascular disease [CVD]) with diabetes and 34 normal control subjects. RESULTS: Plasma vWf (P=0.009), IL-6 (P <0.001), VEGF (P=0.001), and Ang-2 (P=0.001), but not Ang-1 (P=0.635), were higher in diabetic patients with and without CVD than in control subjects. On multivariate analysis, HbA1c was an independent predictor of plasma VEGF (P=0.032) and Ang-2 (P=0.015). Of the 94 patients, a subgroup of 33 patients with and 31 patients without CVD participated in a year of intensified cardiovascular risk management. HbA1c and LDL cholesterol reduced significantly with treatment, along with associated reductions in plasma vWf and VEGF in both groups (P <0.001). Ang-2 decreased (P <0.001) only in patients without CVD. There were no significant changes in plasma IL-6 levels in both groups. CONCLUSIONS: Plasma Ang-2 (but not Ang-1), like VEGF levels, are selectively elevated in patients with diabetes and are associated with indexes of endothelial damage/dysfunction, regardless of vascular disease. Intensive multifactorial intervention is associated with reductions in plasma VEGF, vWf, and (in patients without CVD) Ang-2 levels, possibly reflecting an improved vascular profile with treatment.     

Role of angiogenic growth factors in transplant coronary artery disease

Transplant coronary artery disease (TxCAD) as a manifestation of chronic rejection is a major limitation to long‐term survival of heart transplant recipients. Although the exact molecular and cellular mechanisms contributing to neointimal formation are unknown, it has been generally believed that smooth muscle cells (SMC) of donor origin migrate from the media into the subendothelial layer of the vascular wall, where SMC proliferate and synthesize extracellular matrix resulting in intimal thickening. However, recent observations indicate that hematopoietic and vascular progenitor cells derived from recipient bone marrow may contribute to the arteriosclerotic lesion formation in the coronary arteries of the transplant. On the other hand, studies on postnatal hematopoiesis indicate that angiogenic growth factors such as vascular endothelial growth factor (VEGF) and angiopoietin‐1 (Ang1) may regulate the recruitment of these cells into distant organs. Furthermore, embryonic VEGFR‐2 ⁺ /CD34 ⁺ stem cells may serve as vascular progenitor cells and their differentiation into endothelial cells and SMC may be regulated by VEGF and platelet‐derived growth factor (PDGF), respectively. In this review, we discuss the role of angiogenic growth factors such as VEGF, Ang, and PDGF in the recruitment of hematopoietic and vascular progenitor cells in TxCAD and suggest novel therapies targeted at homing, differentiation and proliferation of these cells in the allograft.     

Association of growth factors with arterial recanalization and clinical outcome in patients with ischemic stroke treated with tPA

Summary. Background: Growth factors (GF) such as vascular endothelial growth factor (VEGF), angiopoietin‐1 (Ang‐1) and granulocyte‐colony stimulating factor (G‐CSF) have been associated with greater efficacy of tissue plasminogen activator (tPA) in experimental studies. Objectives: To study the association of these GF with arterial recanalization and clinical outcome in patients with acute ischemic stroke treated with tPA. Methods: We prospectively studied 79 patients with ischemic stroke attributable to MCA occlusion treated with i.v. tPA within the first 3 h from onset of symptoms. Continuous transcranial color‐coded sonography (TCCS) was performed during the first 2 h after tPA bolus to assess early MCA recanalization. Hemorrhagic transformation (HT) was classified according to ECASS II definitions. Good functional outcome was defined as a Rankin scale score of 0–2 at 90 days. GF levels were determined by ELISA. Results: Mean serum levels of VEGF, G‐CSF and Ang‐1 at baseline were significantly higher in patients with early MCA recanalization (n = 30) (all P < 0.0001). In the multivariate analysis, serum levels of VEGF (OR, 1.03), G‐CSF (OR, 1.02) and Ang‐1 (OR, 1.07) were independently associated with early MCA recanalization (all P < 0.0001). On the other hand, patients with parenchymal hematoma (PH) (n = 20) showed higher levels of Ang‐1 (P < 0.0001). Ang‐1 (OR, 1.12; P < 0.0001) was independently associated with PH, whereas patients with good outcome (n = 38) had higher levels of G‐CSF (P < 0.0001). G‐CSF was independently associated with good outcome (OR, 1.12; P = 0.036). Conclusions: These findings suggest that GF may enhance arterial recanalization in patients with ischemic stroke treated with t‐PA, although they might increase the HT.     

Using angiogenic factors and their soluble receptors to predict organ dysfunction in patients with disseminated intravascular coagulation associated with severe trauma

ABSTRACT: INTRODUCTION: Disseminated intravascular coagulation (DIC) is characterized by the concomitant activation of coagulofibrinolytic disorders and systemic inflammation associated with endothelial dysfunction-induced microvascular permeability. Angiogenic factors, including vascular endothelial growth factor (VEGF), angiopoietin (Ang), and their receptors, play crucial roles in angiogenesis and microvascular permeability. The aim of the study was to assess the relationship between angiogenic factors, their soluble receptors and organ dysfunction associated with DIC after severe trauma. MATERIALS AND METHODS: A total of 57 patients with severe trauma were divided into two subgroups; 30 DIC patients and 27 non-DIC patients. The DIC was diagnosed based on the Japanese Association for Acute Medicine (JAAM) DIC and the International Society on Thrombosis and Haemostasis (ISTH) overt DIC criteria. The serum levels of angiogenic factors were measured at the time of admission (Day 1), Day 3 and Day 5. This study compared levels of these angiogenic factors between the two DIC groups, and evaluated their predictive value for organ dysfunction. RESULTS: DIC patients, especially those with ISTH DIC, showed higher Sequential Organ Failure Assessment (SOFA) scores and lactate levels. There were lower levels of VEGF, Ang1 and the soluble Tie2 in the ISTH DIC patients than the non-DIC patients. The levels of soluble VEGF receptor-1 (sVEGFR1), Ang2 and the Ang2/Ang1 ratio in the ISTH DIC patients were higher than in non-DIC patients. The relationship between the presence of massive transfusion and angiogenic factors indicated the same results. The levels of sVEGFR1, Ang2 and the Ang2/Ang1 ratio correlated with the SOFA scores. In particular, sVEGFR1 and Ang2 were independent predictors of an increase in the SOFA score. The lactate levels independently predicted increases in the levels of sVEGFR1 and Ang2. The decrease in the platelet counts also independently predicted the increase in Ang2 levels in DIC patients. CONCLUSIONS: Angiogenic factors and their soluble receptors, particularly sVEGFR1 and Ang2, are considered to play pivotal roles in the development of organ dysfunction in DIC associated with severe trauma. DIC-induced tissue hypoxia and platelet consumption may play crucial roles in inducing sVEGFR1 and Ang2, and in determining the prognosis of the severity of organ dysfunction.     

Silk fibroin scaffolds loaded with angiogenic genes in adenovirus vectors for tissue regeneration

Vascularization remains a critical challenge in dermal tissue regeneration. In this study, a vascular endothelial growth factor (VEGF165) and angiopoietin‐1 (Ang‐1) dual gene coexpression vector that encoded green fluorescent protein (GFP) was constructed from an arginine–glycine–aspartic acid‐modified adenovirus. Silk fibroin (SF) scaffolds loaded with adenovirus vectors were fabricated by freeze‐drying method. In vitro, the human endothelial‐derived cell line EA.hy926 was infected with adenovirus vectors and then expressed GFP, secreted VEGF165 and Ang‐1, and promoted cell proliferation effectively. The VEGF165 and Ang‐1 genes loaded in the SF scaffolds significantly promoted the formation of abundant microvascular networks in the chick embryo chorioallantoic membrane. In vivo, angiogenic genes loaded in the scaffolds promoted vascularization and collagen deposition in scaffolds, thus effectively accelerating dermal tissue regeneration in a dorsal full‐thickness skin defect wound model in Sprague–Dawley rats. In conclusion, SF scaffolds loaded with arginine–glycine–aspartic acid‐modified adenovirus vectors encoding VEGF165 and Ang‐1 could stimulate the formation of vascular networks through the effective expression of target genes in vascular endothelial cells, thereby accelerating the regeneration of dermal tissue.     

Prognostic value of interleukin-6, plasma viscosity, fibrinogen, von Willebrand factor, tissue factor and vascular endothelial growth factor levels in congestive heart failure

BACKGROUND: Congestive heart failure (CHF) carries a poor prognosis with a high mortality rate, frequent hospitalizations and increased risk of thrombotic complications such as stroke. Cytokines may contribute to the progression and prothrombotic state of CHF, including the pro-inflammatory interleukin-6 (IL-6) and the pro-angiogenic vascular endothelial growth factor (VEGF), both of which are raised in CHF. The procoagulant properties of both cytokines may be mediated via tissue factor (TF), a potent clotting activator. We hypothesized that plasma levels of these markers, as well as levels of plasma viscosity, fibrinogen, soluble P-selectin and von Willebrand factor (markers of abnormal rheology, clotting, platelet activation, and endothelial damage, respectively) will be useful in predicting morbidity and mortality in chronic stable CHF. METHODS AND RESULTS: One hundred and twenty consecutive out-patients with chronic stable CHF (92 males; mean [SD] age 64 [11] years, mean [SD] left ventricular ejection fraction of 29 [6]%) were recruited and followed for 2 years during which 42 patients reached a clinical end-point of all-cause mortality and cardiovascular hospitalizations, including stroke and myocardial infarction. Plasma IL-6 (P=0.003) and TF (P=0.013) levels, but not other research indices, were higher in those who suffered events compared with those without events. Predictors of end-points were high (> or =median) TF (P=0.011), and IL-6 (P=0.023) levels, as well as the lowest quartile of a left ventricular ejection fraction (P=0.007). A strong correlation was present between TF and IL-6 levels (r=0.59; P<0.0001) and with VEGF levels (r=0.43; P<0.0001). CONCLUSION: IL-6 and TF are predictors of poor prognosis in chronic CHF, raising the hypothesis that IL-6 may contribute to the progression and thrombotic complications of CHF via its actions on TF expression. Although VEGF did not independently predict outcome in chronic CHF, the possibility arises that it may act with IL-6 to induce TF expression.     

Heparin promotes soluble VEGF receptor expression in human placental villi to impair endothelial VEGF signaling

Summary. Background: Severe preeclampsia is characterized by hypertension, renal injury and placental dysfunction. Prothrombotic disorders are discovered in 10–20% of women with preeclampsia, providing the rationale for prescribing low‐molecular‐weight heparin (LMWH) in future pregnancies. Heparin has diverse molecular actions and appears to reduce the recurrence risk of preeclampsia in women without prothrombotic disorders. The placenta‐derived anti‐angiogenic splice‐variant protein soluble vascular endothelial growth factor (VEGF) receptor‐1 (sFLT1) is strongly implicated in the pathogenesis of the underlying endothelial dysfunction. As the placental syncytiotrophoblast is the principal source of sFLT1, we tested the hypothesis that heparin suppresses placental sFLT1 secretion. Methods and Results: First trimester placental villi exposed to LMWH (0.25–25 IU mL^?1) in an in vitro explant model significantly increased the expression and release of sFLT1 by the syncytiotrophoblast into culture media, reducing phosphorylation of FLT1 and KDR receptors in cultured human umbilical vein endothelial cells. This response was significantly diminished in placental villi from healthy term pregnancies. Placental villi from severely preeclamptic pregnancies had a higher baseline sFLT1 release, compared with first trimester placental villi and did not respond to LMWH treatment. LMWH promoted villous cytotrophoblast proliferation (BrdU incorporation) and impaired syncytial fusion‐differentiation, causing syncytiotrophoblast apoptosis (by caspase 3&7 activity and TUNEL staining) and necrosis (ADP/ATP ratio). Conclusion: LMWH promotes sFLT1 synthesis and release from first trimester placental villi in a manner similar to that of severely preeclamptic placental villi, which antagonizes VEGF signaling in endothelial cells. These effects in part are mediated by an interaction between heparin and the cytotrophoblasts that regenerates the overlying syncytiotrophoblast responsible for sFLT1 secretion into the maternal blood.     

Does low angiopoietin-1 predict adverse outcome in sepsis?

Endothelial injury has emerged as a crucial early event in the pathogenesis of microcirculatory dysfunction, capillary leakage and multiorgan dysfunction syndrome. The endothelial-specific angiopoietin (Ang)/Tie2 ligand-receptor system has been identified recently as a nonredundant regulator of endothelial responsiveness. Ang-1 is a Tie2 agonist and promotes endothelial stabilization and quiescence, whereas Ang-2 is a Tie2 antagonist and promotes endothelial activation, destabilization, and inflammation. While the mediator function of both Ang-1 and Ang-2 has been well established in preclinical research, only Ang-2 has been identified as a clinically useful biomarker in the critical care arena. In the previous issue of Critical Care, Mankhambo and colleagues report on angiogenic factors in Malawian children with severe bacterial infection. Among those children, diminished levels of the vessel-protective factor Ang-1 remained a significant predictor of outcome after multivariate adjustment. Whether low Ang-1 represents an important risk factor of adverse outcome in critically ill adults remains to be seen.     

Vascular defects in gain‐of‐function fps/fes transgenic mice correlate with PDGF‐ and VEGF‐induced activation of mutant Fps/Fes kinase in endothelial cells

Summary. Background: Fps/Fes is a cytoplasmic tyrosine kinase that is abundantly expressed in the myeloid, endothelial, epithelial, neuronal and platelet lineages. Genetic manipulation in mice has uncovered potential roles for this kinase in hematopoiesis, innate immunity, inflammation and angiogenesis. Objective: We have utilized a genetic approach to explore the role of Fps/Fes in angiogenesis. Methods: A hypervascular line of mice generated by expression of a ‘gain‐of‐function’ human fps/fes transgene (fps^MF) encoding a myristoylated variant of Fps (MFps) was used in these studies. The hypervascular phenotype of this line was extensively characterized by intravital microscopy and biochemical approaches. Results: fps^MF mice exhibited 1.6–1.7‐fold increases in vascularity which was attributable to increases in the number of secondary vessels. Vessels were larger, exhibited varicosities and disorganized patterning, and were found to have defects in histamine‐induced permeability. Biochemical characterization of endothelial cell (EC) lines derived from fps^MF mice revealed that MFps was hypersensitive to activation by vascular endothelial growth factor (VEGF) and platelet‐derived growth factor (PDGF). Conclusions: MFps mediates enhanced sensitization to VEGF and PDGF signaling in ECs. We propose that this hypersensitization contributes to excessive angiogenic signaling and that this underlies the observed hypervascular phenotype of fps^MF mice. These phenotypes recapitulate important aspects of the vascular defects observed in both VEGF and angiopoietin‐1 transgenic mice. The fps/fes proto‐oncogene product therefore represents a novel player in the regulation of angiogenesis, and the fps^MF line of mice constitutes a unique new murine model for the study of this process.     

Factor VII –323 decanucleotide D/I polymorphism in atrial fibrillation: Implications for the prothrombotic state and stroke risk

There are limited data on the influence of genetic polymorphisms in atrial fibrillation (AF) stroke risk. We hypothesized that a functional haemostatic polymorphism, that is, the factor VII ?323 Del/Ins polymorphism, would influence the prothrombotic state associated with AF, as well as stroke risk. Other functional polymorphisms were also tested.

Methods. We performed a cross-sectional study of 119 AF patients, who were compared to 96 patients with stroke secondary to AF. In the first patient group, we analysed plasma prothrombin fragment 1+2 levels (F1+2, an index of thrombin generation) to reflect the prothrombotic state of AF.

Results. AF patients carrying the ?323 Ins allele had lower plasma F1+2 levels (P=0.015). After multivariate analysis adjusted by age, sex and clinical risk factors, advanced age and 807C/T polymorphism of glycoprotein Ia (GPIa) gene were associated with higher risk of ischaemic stroke (OR: 1.06; P=0.003 and OR: 1.91; P=0.025), whilst FVII Ins ?323 allele was associated with lower stroke risk (OR: 0.41; P=0.017).

Conclusion. FVII ?323 Ins allele may modulate the prothrombotic state associated with AF. Despite the small sample size, we found that FVII Ins ?323 allele could be associated with a lower stroke risk in AF, whereas the 807C/T polymorphism may increase the risk.     

Enhanced monocyte expression of tissue factor by oxidative stress in patients with antiphospholipid antibodies: effect of antioxidant treatment

Summary. In a first study, we performed a cross‐sectional analysis of urinary excretion of isoprostanes, IPF_2α‐III and _VI, and monocyte tissue factor (TF) antigen and activity between 11 antiphospholipid (APL) antibody‐positive patients and 13 APL negative subjects. In a second study, 11 APL positive patients were randomly supplemented either with (n = 6) or without (n = 5) antioxidants (vitamin E at 900 IU day^?1, vitamin C at 2000 mg day^?1) for 6 weeks. In a third study, TF and superoxide anion were measured in human monocytes incubated with anti‐β₂ glycoprotein 1 (β₂GP₁) or control IgG, either with or without vitamin E. APL‐positive patients had higher values of isoprostanes (P < 0.05) and monocyte TF antigen (P = 0.001) and activity (P = 0.0001) than APL‐negative subjects. Only in APL positive patients did monocyte TF antigen correlate significantly with IPF_2α‐III (rho 0.79; P < 0.003) and IPF_2α‐VI (rho = 0.87; P < 0.0001). In patients who received antioxidant supplementation, we found a significant decrease of isoprostanes (P < 0.05) and monocyte TF antigen (P < 0.01) and activity (P < 0.007). In vitro experiments demonstrated that anti‐β₂GP₁ antibodies dose‐dependently enhanced the monocyte production of the superoxide anion and TF, which were significantly inhibited by vitamin E. This study demonstrates that in APL‐positive patients, oxidative stress contributes to activate the clotting system via over‐expression of monocyte TF. We suggest that anti‐β₂GP₁ antibodies could play a pivotal role by enhancing the monocyte production of oxygen free radicals.