Upstream thrombus growth impairs downstream thrombogenesis in non-anticoagulated blood: effect of procoagulant artery subendothelium and non-procoagulant collagen

In the present experiments we have investigated the influence of wall shear rate and axial position on platelet and fibrin deposition which results when flowing human non-anticoagulated blood is exposed to either non-procoagulant fibrillar collagen (human type III) or procoagulant subendothelium (rabbit aorta). Platelet adhesion, thrombus volume and fibrin deposition were morphometrically evaluated at axial positions of 1 and 13 mm following perfusions for 5 min at shear rates of 100, 650 and 2,600 s-1. An axially-dependent decrease of platelet adhesion (34-57%, p less than 0.01-0.05) and thrombus volume (57-80%, p less than 0.05) was observed on collagen at all shear rates. On subendothelium, an axially-dependent decrease was observed for platelet adhesion only at 100 s-1 (29%; p less than 0.01) and for thrombus volume at shear rates of 650 s-1 and above (49-58%, p less than 0.01). Deposition of fibrin on subendothelium was axially decreased (16-42%, p less than 0.05) at all shear rates, while no significant axial differences were seen on collagen. However, substantially more fibrin was deposited on the subendothelium (p less than 0.05), and the upstream platelet adhesion and thrombus volume were lower than on collagen (p less than 0.05) at 100 s-1 and 650 s-1. The axially-dependent phenomena on the two surfaces are consistent with the concept of rapid-growing upstream thrombi which deplete the blood layer streaming adjacent ot the surface of platelets, leading to decreased platelet deposition further downstream.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective venous thrombolysis with the nipple-balloon catheter: comparative evaluation in vivo.

PURPOSE: To compare in an animal model of deep vein thrombosis, an intramural drug delivery catheter, the nipple-balloon catheter, with an occlusion balloon-infusion guide wire system. MATERIALS AND METHODS: Ten juvenile pigs were used for the study. Deep vein thrombosis was induced in both hind limbs by using a previously described technique. Heparin was administered 30 minutes later (2,500 IU intravenously) and bilateral thrombolysis was attempted with use of 8 mg of alteplase as a 0.25 mg/mL solution containing heparin 50 IU/mL (n = 10) and sodium/meglumine ioxaglate 40 mgI2/mL (n = 5). In one limb, the external iliac vein was endoluminally occluded, and 0.8 mL of alteplase was administered every 3 minutes through a multisideport infusion wire placed coaxially through the balloon catheter. On the other side, a nipple-balloon catheter was used: alteplase was injected as two 0.4-mL aliquots every 3 minutes in overlapping segments of the vessel. Blood samples were taken at predetermined intervals to determine the partial thromboplastin time and plasma fibrinogen concentration. At autopsy, the thrombus mass in the iliofemoral veins was measured, and the extent of residual thrombosis in the venous tributaries was graded at four sites. The heart and the lungs were also examined for thromboemboli (n = 5). Venous specimens were then subjected to X-ray fluorescence spectrometry to determine iodine content (n = 5). RESULTS: Bilateral thrombolysis could be successfully completed in all animals. No procedural problem associated with the use of the nipple-balloon catheter was encountered. The mass of residual thrombus in the axial veins was significantly lower in this group (P = .005). The drug delivery system used did not appreciably influence thrombolysis in the tributaries. Signs of macroscopic damage to the veins were not observed in any animal. None of the venous specimens had detectable levels of iodine. Small thromboemboli were found in the pulmonary circulation in three of five animals. Fibrinogen levels did not decrease during the procedure. CONCLUSIONS: The significantly lower residual thrombus burden associated with use of the nipple-balloon catheter suggests that the device may have the potential to be an effective delivery system for selective thrombolysis in veins.     

Factor VIII-von Willebrand factor requires calcium for facilitation of platelet adherence

The role of divalent cations in platelet adherence to deendothelialized human arteries in flowing blood was investigated in an annular perfusion chamber. Spreading of platelets on the subendothelium was impaired below 30 microM of free Ca2+ ions (Ca2+). When Ca2+ was replaced by Mg2+, adherence was unchanged in perfusates without exogenous factor VIII-von Willebrand factor (FVIII-vWF), but the ability of FVIII-vWF to support platelet adherence was lost. Binding of FVIII-vWF to the vessel wall was independent of divalent cations, but bound FVIII-vWF was only able to mediate adherence after exposure to Ca2+. Pretreatment of FVIII-vWF with the calcium chelator EGTA (10 mM) resulted in loss of the ability to facilitate platelet adherence, while the ristocetin cofactor activity remained intact. Full restoration of the ability to mediate platelet adherence could only be obtained by prolonged dialysis against Ca2+ in the millimolar range. These data indicate that divalent cations have at least two separate roles to play in supporting platelet adherence: (1) platelet spreading on the subendothelium requires Ca2+ or Mg2+; (2) FVIII-vWF should be exposed to Ca2+ to obtain its optimal biologic activity in supporting platelet adherence.     

Nonfractionated Heparin Fails to Inhibit Arterial Thrombosis in a Human Ex Vivo Thrombosis Model

−1 for 1 to 4 min. Platelet deposition was determined using immunoenzymatic techniques to quantify P-selectine, a platelet membrane receptor, in thrombi. Fibrin deposition was determined by quantifying fibrin degradation products released after application of plasmin (D-dimers). Heparin was injected into the blood flow through a blender port system located between the venous puncture site and perfusion chamber. The results of the study showed that in a human ex vivo model, formation of arterial thrombus on two thrombogenic surfaces (tissue factor and collagen) is not inhibited by nonfractionated heparin.     

Effect of radiologic contrast media on cell volume regulatory mechanisms in human red blood cells

RATIONALE AND OBJECTIVES: The authors performed this study to evaluate cell volume regulation in human red blood cells (RBCs) after incubation in solutions of three contrast media: iohexol (830 mOsm), ioxaglate (520 mOsm), and iodixanol (300 mOsm). MATERIALS AND METHODS: Whole blood sampled from six healthy subjects was exposed to Ringer solutions containing 25% or 5% vol/vol iohexol (final osmolality, 440 or 340 mOsm, respectively), ioxaglate (final osmolality, 395 or 335 mOsm, respectively), iodixanol (final osmolality, 330 or 315 mOsm, respectively), or NaCl (control solutions with the same osmolality as that of the contrast media). In some experiments, control RBCs were subjected to a hyposmotic solution (100 mOsm). RBC volumes were obtained with a Coulter counter. RESULTS: The RBCs showed normal regulatory cell shrinkage after hyposmotically induced swelling. All 25% vol/vol contrast material solutions and their control solutions induced RBC shrinkage (range, 6% +/- 1 [standard error] to 22% +/- 3). The same was true for cells exposed to 5% vol/vol contrast material (range, 4% +/- 1 to 7% +/- 1). The shrinkage phase was followed by cell swelling (10% +/- 2 to 20% +/- 2 for 25% contrast material and their control solutions and 8% +/- 1 to 15% +/- 2 for 5% contrast material and their control solutions). No contrast material-exposed RBCs increased their volumes to the level reached with their control solutions. CONCLUSION: RBCs exposed to hyperosmotic iohexol, ioxaglate, or iodixanol solutions shrank and then swelled. The degree of shrinkage and subsequent swelling could not be explained simply with the osmolality of the test solutions. Physicochemical properties of the contrast media must be involved, putatively affecting electrolyte fluxes over the RBC membrane. Possible targets of these effects are the K+/Cl- symporter, K+ channels, and the Na+/K+/Cl- symporter.     

Superior efficacy of clopidogrel plus acetylsalicylic acid compared with extended-release dipyridamole plus acetylsalicylic acid in preventing arterial thrombogenesis in healthy volunteers

INTRODUCTION: Recent ex vivo platelet aggregometry data indicate that clopidogrel 75 mg/day plus acetylsalicylic acid (ASA) 75 mg/day is a more potent antiplatelet regimen than the marketed combination of dipyridamole+ASA. The present study was designed to assess the antithrombotic effect of both dual antiplatelet regimens using a human ex vivo model of arterial thrombosis. MATERIALS AND METHODS: This was a randomized, double-blind, placebo-controlled, crossover study. During two 10-day treatment periods separated by a 14-day washout period, 23 healthy male volunteers received once-daily clopidogrel 75 mg plus acetylsalicylic acid 75 mg, or twice-daily extended-release dipyridamole 200 mg plus acetylsalicylic acid 25 mg. Assessments were made at baseline and on Day 10 of each period. Arterial thrombus formation was induced ex vivo by exposing a collagen-coated surface in a parallel-plate perfusion chamber to native blood for 3 min (arterial wall shear rate 2600 s(-1)). Total platelet and fibrin deposition was determined by immunoenzymatic methods. RESULTS: Compared with baseline values, the mean inhibition of total platelet deposition was 63.9+/-5.9% with clopidogrel plus acetylsalicylic acid, compared with 18.4+/-5.6% for extended-release dipyridamole plus acetylsalicylic acid (67% reduction; 95% CI, 49-79%; p<0.0001). Corresponding figures for fibrin deposition were 64.9+/-4.8% and 18.3+/-9.7%, respectively (58% reduction; 95% CI, 45-67%; p<0.0001). Both treatments were well tolerated. CONCLUSIONS: Clopidogrel plus acetylsalicylic acid showed significantly superior antithrombotic efficacy compared with extended-release dipyridamole plus acetylsalicylic acid in preventing arterial thrombogenesis in humans.     

Angiogenesis-independent tumor growth mediated by stem-like cancer cells

In this work, highly infiltrative brain tumors with a stem-like phenotype were established by xenotransplantation of human brain tumors in immunodeficient nude rats. These tumors coopted the host vasculature and presented as an aggressive disease without signs of angiogenesis. The malignant cells expressed neural stem cell markers, showed a migratory behavior similar to normal human neural stem cells, and gave rise to tumors in vivo after regrafting. Serial passages in animals gradually transformed the tumors into an angiogenesis-dependent phenotype. This process was characterized by a reduction in stem cells markers. Gene expression profiling combined with high throughput immunoblotting analyses of the angiogenic and nonangiogenic tumors identified distinct signaling networks in the two phenotypes. Furthermore, proinvasive genes were up-regulated and angiogenesis signaling genes were down-regulated in the stem-like tumors. In contrast, proinvasive genes were down-regulated in the angiogenesis-dependent tumors derived from the stem-like tumors. The described angiogenesis-independent tumor growth and the uncoupling of invasion and angiogenesis, represented by the stem-like cancer cells and the cells derived from them, respectively, point at two completely independent mechanisms that drive tumor progression. This article underlines the need for developing therapies that specifically target the stem-like cell pools in tumors.     

A perfusion chamber developed to investigate platelet interaction in flowing blood with human vessel wall cells, their extracellular matrix, and purified components

A flat perfusion chamber was developed to study the interaction of blood platelets in flowing blood with cultured human vessel wall cells, their connective tissue matrix, and isolated connective tissue components at defined shear rate conditions. A cover slip covered with endothelial cells or extracellular matrix components was introduced into the chamber. Laser-Doppler velocimetry showed a symmetrical flow profile at flow rates between 50 and 150 ml/min (wall shear rate 300 to 1100 sec-1). Platelet deposition was estimated by using blood platelets labeled with indium-111 or by a morphometric method. Blood platelets did not adhere to endothelial cells at wall shear rates of 765 sec-1 and the endothelial cells remained attached for at least 10 min of perfusion. In preconfluent cultures of endothelial cells, blood platelets adhered to extracellular material in areas between the cells. Removal of endothelial cells by treatment with 0.5% Triton X-100 induced increased platelet adherence with a preference for certain, as yet unidentified, fibrillar structures of the extracellular matrix. Platelet adherence to equine collagen was also studied after coating the cover slips by spraying of small collagen droplets followed by air drying. Platelet adherence and the subsequent platelet aggregate formation occurred predominantly along visible collagen fibers. These studies showed that this perfusion chamber has a laminar and symmetrical flow allowing qualitative and quantitative investigation of platelet interaction with endothelial cells, their extracellular matrix, and pure connective tissue components. A variety of wall shear rates and exposure times can be applied at controlled conditions without removing cells or extracellular material.     

Bevacizumab treatment for human glioblastoma. Can it induce cognitive impairment?

Recent results from 2 double-blind, placebo-controlled phase III trials (RTOG 0825) and (AVAglio) for first-line treatment of glioblastoma patients with the VEGF antibody bevacizumab, showed similar results, related to overall and progression-free survival. The RTOG 0825 trial indicated, opposed to the AVAglio trial, that patients treated with bevacizumab showed a decline in global neurocognitive function compared to untreated patients, -a decline that was most obvious after prolonged treatment. At present, there is a considerably controversy related to these observations. In the present work we point at the possibility that bevacizumab treatment of the normal brain can reduce synaptic plasticity in the hippocampus. We believe that such a phenomenon may partly explain the reduced cognitive function observed in patients in the RTOG 0825 trial. Since the same effects were not clearly defined in the AVAglio trial, further studies on putative neurocognitive effects after bevacizumab treatment are warranted.Dear Editor,The vascular endothelial growth factor (VEGF) inhibitor bevacizumab (Avastin) has since 2009 been extensively used in the clinic for the treatment of recurrent glioblastoma (GBM). Recently, results from two double-blind, placebo-controlled phase III trials, one publicly supported by the National Cancer Institute (NCI), conducted by the Radiation Therapy Oncology Group¹ (RTOG 0825), and one industry sponsored trial² (AVAglio) were released (The 2013 ASCO Annual meeting). The results from both trials show that bevacizumab treatment does not increase overall survival (OS) in the patient treatment group, whereas progression-free survival was improved in the bevacizumab arm, yet with some differences between the two trials. As a part of the results from the RTOG 0825 trial it was reported that patients in the bevacizumab arm showed less quality of life (QOL) and a worse symptom burden related to neurocognitive function^1,2 an observation that was less recognized in the AVAglio trial. In the RTOG 0825 trial, 507 patients were evaluated at diagnosis and at intervals throughout bevacizumab treatment. Objective tests of cognitive function and subjective assessments of symptoms and quality of life were performed during periods were the tumors were not obviously progressing. Longitudinal analyses indicated that patients treated with bevacizumab showed a more pronounced decline in global neurocognitive function compared to untreated patients, a decline that was most obvious after prolonged treatment.Based on these observations, there is an obvious need for further studies adding to the questions whether bevacizumab induces a putative cognitive impairment in the normal brain or whether such an impairment is a result of treatment effects on the tumor tissue.It is well known that VEGF-A signaling modulates both vascular and neuronal behavior in the central nervous system (CNS). For instance it has been shown that VEGF-A can increase neurite number, length and size in the absence of glia cells, indicating an essential role in neuronal function.^3–5 VEGF-A is also a potent survival factor for many neuronal populations^5,6 and may provide neuroprotection from hypoxia^7,8 and mechanical trauma⁹ which represent important environmental factors induced by progressive tumor growth. In the normal adult brain VEGF is expressed in a region specific manner that is incompatible with angiogenic growth¹⁰ indicating angiogenesis and perfusion independent functions of VEGF in the CNS. In particular, VEGF is expressed in the choroid plexus, in the olfactory bulb, in the cortex (pyramidal neurons), in the cerebellum (Purkinje cells) and in the hippocampus (by CA1 pyramidal neurons).¹⁰ The hippocampal regions known as CA1, CA3 and Dentate gyrus (DG) play an important role in spatial learning and short term memory function. At present there is considerable consensus that VEGF is not essential for maintaining basal neurogenesis but may be important in maintaining homeostatic functions in the CNS.¹⁰ In the hippocampus, it has been shown that VEGF overexpression can augment learning and memory whereas loss of function impairs this effect.^11,12Synaptic plasticity (SP) describes changes in synaptic strength that among others are important in learning and memory. SP is orchestrated by the amount of neurotransmitters available, the synaptic density and how effectively the cells respond to neurotransmitters. It is well known that a complex web of intracellular signaling pathways mediates SP. An informative study linking VEGF to synaptic plasticity was performed using long-term potentiation (LTP), which represents a method to determine dynamic plasticity changes in the hippocampus. This method measures the increase in synaptic response following potentiating pulses of electrical stimuli that are sustained at a level above the baseline response for hours or longer.¹³ In the context of VEGF, hippocampal slice cultures, as well as in vivo experiments have shown VEGF to increase LTP in CA1 pyramidal cells and that the absence of VEGF abrogates this effect.^11,14 It is therefore highly likely that reduced VEGF levels in the CNS may affect neuronal populations. A key question in this context is the source of VEGF in the normal brain. In the brain parenchyma, co-expression studies have shown that both astrocytes and neurons can secrete VEGF, that is up-regulated by hypoxia inducible factor (HIF1) target genes.^15–17We have recently shown that vascular remodeling induced by bevacizumab treatment leads to a more hypoxic tumor microenvironment which favors metabolic changes in GBMs toward glycolysis.¹⁸ This study indicates that induced hypoxia, mediated by bevacizumab treatment, may also lead to an induction of VEGF production in neural cell populations to maintain homeostasis. It is therefore likely that the presence of the neutralizing antibody bevacizumab, during treatment abrogates the neuroprotective effects of VEGF.As a part of our bevacizumab treatment study¹⁸ we performed a number of control experiments where we treated normal non-tumor bearing rats with bevacizumab (10 mg/kg once a week during a 3-week period). We then performed gene expression analyses and observed significant changes in gene expression in the treatment group compared to the control group (3074 differentially expressed genes at a FDR of 5%) (Fig. 1A). Based on GO terms, not surprisingly, genes important for endothelial function and development were observed in the treatment group, but also a deregulation of genes involved in CNS function and development (Fig. 1A). More interesting, we then performed a highly standardized LTP experiment, comparing the bevacizumab treatment group with the control group. As seen in Fig. 1B, treatment caused a strong reduction in LTP compared to the controls animals, strongly implying a reduced neurocognitive function after bevacizumab treatment. Open in a separate windowFig. 1.(A) Gene expression data from four rats in each of treated (bevacizumab) and control (vehicle) groups. Whole genome gene expression data was obtained using RNA extracted from rat brains of animals in the 2 groups. The Agilent 4 × 44k v3 rat Gene Expression Arrays were used. Principal Component Analysis (PCA) was carried out using Partek^® and differentially expressed genes were obtained using Limma (Linear Models for Microarray Data in R) PCA shows that 2 main clusters are obtained (Ctrl and Treat). A functional analysis based on the list of differentially expressed genes was performed using Ingenuity Pathway analysis (IPA). The table shows a selection of gene ontology (GO) terms that are highly enriched in Avastin treated animals (right panel). (B) Hippocampal LTP recordings obtained from 6 rats (treatment group receiving 10 mg/kg bevacizumab twice a week during a 3-week period) and from 6 control rats (receiving sodium chloride) showing a significant LTP reduction in the bevacizumab treated group. Briefly, a bipolar concentric stimulating electrode was placed in the perforant path and a recording electrode was inserted into the dentate gyrus of the dorsal hippocampus according to stereotactic coordinates as described in.¹⁹ Evoked responses were amplified, filtered at 1 Hz to 1 kHz, and stored for later analysis. Input–output relations was examined by using three stimulus intensities (1.5, 3, and 4.5V). Paired-pulse responses were measured at three interstimulus intervals (15, 30, and 60 ms). LTP was induced by applying high frequency stimulation (HFS) of 5 trains of 8 0.4-ms, 400-Hz pulses of 1.5V. Ten measurements were taken every 5 min and LTP was computed as the change in the evoked responses measured during 60 min. after HFS in comparison with pre-HFS responses. 2-way Anova analysis using Matlab 8.2 (MathWorks Inc, Natick, MA, USA), using the treatment and time as factors of variation. The treatment significantly affected the LTP response (P value <10⁻⁵) throughout the whole duration of the LTP experiment. (ANOVA P < .02).In conclusion, in light recent clinical trials and supporting the observations made in the RTOG 0825 trial,¹ the relatively simple experiments performed here, strongly support the notion that bevacizumab can cause cognitive impairment. The fact that bevacizumab will reduce circulating as well as local VEGF levels in the brain may lead to a reduced accessibility of this growth factor to neurons that can lead to cognitive impairment. A central question, that warrants further studies, is if this impairment is reversible or not. In our mind this question should be addressed by in depth neuro-cognitive studies of cancer patients treated with bevacizumab for other malignancies (eg colorectal cancer).