Vascular Biology
PP5.1 Animal and Experimental Models
Clopidogrel improves endothelial function and NO bioavailability in congestive heart
Vogt C1, Fraccarollo D1, Ertl G1, Bauersachs J1, Schäfer A1
1Medizinische Universitätsklinik Würzburg, Germany
Background: Treatment of patients with coronary artery disease with clopidogrel
not only inhibits platelet activation, but also improves endothelial function and NO
bioavailability. Similarl to coronary artery disease, patients with chronic congestive
heart failure (CHF) display endothelial dysfunction and increased platelet activa-
tion. Using the experimental CHF model following chronic myocardial infarction
(MI) in rats we investigated, whether treatment with clopidogrel positively modi-
fies endothelial function in CHF.
Methods: MI was induced in helthy male Wistar rats by ligation of the left anterior
descending coronary artery. After 8 weeks, animals were randomized to placebo
or the platelet P2Y12 receptor antagonist clopidogrel (5 mg/kg twice dayly, given
Congress Program: 53rd Annual Meeting - Society of Thrombosis and Haemostasis Research Tagungsprogramm: 53. Jahrestagung der Gesellschaft für Thrombose- und Hämostaseforschung e.V. - GTH by gavage) for another 2 weeks. Afterwards, endothelial function was assessed in CHF45; Sham: 34, p<0.01). Furthermore, fractalkine desensitized platelets to the isolated aortic rings in organ bath experiments.
endogenous platelet inhibitors prostacyclin and nitric oxide, whose bioactivity was Results: Endothelium-dependent, acetylcholine-induced vasorelaxation was sig-
nificantly attenuated in CHF rats (EC50: 179.5±19.9 nmol/l, Rmax: 63.5±5.5 %) Conclusion: We observed increased serum fractalkine levels and platelet expres-
compared to sham-operated animals (EC50: 51.5±19.5 nmol/l, Rmax: 93.4±2.6 %, sion of the fractalkine receptor CX3CR1 in heart failure. Fractalkine desensitizes p<0.001), which was significantly improved by treatment with clopidogrel (EC50: platelets to endogenous platelet inhibitors and contributes to impaired clopidogrel 40.4±4.6 nmol/l, p<0.001; Rmax: 99.5±0.3 %, p<0.001). Basal NO bioavailability, which was determined indirectly by additional vasoconstriction of phenyleph-rine-preconstricted aortic rings following addition of the NO synthase-inhibitor Nù-Nitro-L-arginine, was significantly impaired in CHF rats (54.7±3.2 % of maxi- Plasma ADAMTS13 activities in non-human primates and rabbits do not change upon
mum phenylephrine contraction, p<0.001) compared to sham-operated animals administration of human recombinant VWF
(103.9±8.3 % of maximum phenylephrine contraction) and was improved by clopi-dogrel (76.7±2.7 % of maximum phenylephrine contraction, p<0.001).
Varadi K1, Rottensteiner H1, Muchitsch E1, Weber A1, Gritsch H1, Turecek P1, Ehrlich H1, Schwarz H1 Conclusion: Clopidogrel improved endothelial function and NO bioavailability in
rats with CHF.This mechanism might at least partially contribute to the vasoprotec- Objectives: Von Willebrand factor (VWF) is composed of a series of multimers,
the sizes of which are regulated by the plasma metalloprotease ADAMTS13. Tran-sient increases in VWF levels, triggered for instance by DDAVP treatment, were reported to result in a decrease in ADAMTS13 activity, possibly due to its exhaus- New insight into the function of VKORC1L1
tion upon exposure to an excess of substrate. Thus, treating von Willebrand dis-ease type 3 patients with recombinant human von Willebrand factor (rVWF) might Westhofen P1, Watzka M1, Marinova M1, Hass M1, Oldenburg J1 1Uniklinik Bonn; Institut für Exp. Hämatologie und Transfusionsmedizin, Bonn, Germany Design and Methods: The effect of administration of high doses of rVWF on endog-
Objectives: VKORC1L1 is a member of enzymes that are present in vertebrates,
enous ADAMTS13 levels was tested in animal models. Various doses of human plants and bacteria. Furthermore, it is the isozyme of VKORC1 that is known to rVWF (300, 600, and 1200 RCo IU/kg BW) were injected into rabbits and cyn- reduce vitamin-K-epoxide to vitamin-K-quinone in the vitamin K cycle. Herein, we omolgus monkeys, plasma samples were collected at a range of time points, and investigated the role of VKORC1L1 in intracellular antioxidation.
ADAMTS13 activity (FRETS assay) and VWF:Ag were determined. ADAMTS13- Design and Methods: We measured VKOR enzymatic activity and corresponding
specific cleavage products were detected by immunoblot analysis.
expression of VKORC1L1 mRNA from hydrogen peroxide treated HEK cells.
Results: VWF antigen rose sharply in a dose-dependent manner (~25 IU/ml
Activity was assayed by determination of vitamin-K1-epoxide to quinone reduc- VWF:Ag for the highest dose, 15 min after injection) and then declined gradu- tion by HPLC. Transcription rate of VKORC1L1 was analysed by real-time PCR ally (~7 IU/ml VWF:Ag for the highest dose, 18 hours after injection). By con- with specific probes in reference to PBGD as housekeeping gene. Localisation was trast, the ADAMTS13 activity did not show relevant changes throughout the entire performed by co-expression of c-terminal eGFP-tagged VKORC1L1 and com- test period in the rabbit or in the monkey samples. Both rabbit and cynomolgus mercial fluorescent vectors for different subcellular components by fluorescence ADAMTS13 recognized human rVWF as the specific cleavage products were detectable at all doses administered.
Results: Examination of VKORC1L1 response to peroxide treatment revealed a
Conclusions: The animal studies clearly indicate that an excess of intravenously
five-fold increased mRNA-level with a maximum after 40 minutes. As expected, administered rVWF leading to supraphysiological levels does not exhaust VKOR activity was enhanced after induction, too. Furthermore, about 250 % rise in activity sustained at 120 minutes corresponds well to the increase of expressionafter exposure to hydrogen peroxide. Co-expression of labelled VKORC1L1 shows a localisation of VKORC1L1 in the same cellular component like VKORC1, the Measurement of von Willebrand Factor (VWF) function under physiological flow
Conclusion: Rising transcription rate of VKORC1L1 and postponed increased
VKOR activity as reaction to oxidative stress indicates a connection to cellular
Fuchs B1, Budde U2, Fisseau C1, Schulz A3, Kannicht C1 defence against free radicals. The localisation of VKORC1L1 in the endoplasmatic 1Octapharma PPGmbH, Berlin, Germany 2AescuLabor Hamburg GmbH, 3Freie Universität Berlin, reticulum suggests a radical scavenge mechanism to protect this cellular component against oxidative damage caused by oxygen radicals. These underline results our Objectives: VWF is essential for primary haemostasis. Binding of VWF to exposed
hypothesis of an antioxidant function of VKORC1L1.
extracellular matrix components in injured vessel walls leads to platelet tethering,activation and adhesion. At low shear conditions, platelets are able to bind directly to collagen, but under high flow present in human arterial circulation, the presence Fractalkine promotes platelet activation and vascular dysfunction in chronic heart
of plasmatic VWF is essential to achieve stable platelet adhesion. We evaluated the failure: a link to impaired Clopidogrel responsiveness
binding of VWF to immobilized collagen I or III at various shear rates, qualified Schäfer A1, Schulz C2, Fraccarollo D1, Massberg S2, Bauersachs J1 the collagen-bound VWF and analyzed VWF-mediated platelet binding using an in Medizinische Universitätsklinik Würzburg, 2Deutsches Herzzentrum München, Germany Design and Methods: Experiments were performed using physiological concentra-
Background: Endothelial dysfunction and enhanced platelet reactivity in chronic
tions of VWF, erythrocytes and platelets in a flow-chamber (µ-slide VI, ibidi®, Ger- heart failure (CHF) contribute to reduced prognosis. CHF patients display an many) coated with collagen I or III. Binding of Wilate® (Octapharma PPGmbH, impaired response to clopidogrel. We investigated whether fractalkine is linked Austria) was tested applying physiological low to high shear rates. Collagen-bound to impaired clopidogrel responsiveness in patients with CHF. In CHF rats, the VWF was qualified via antibody detection and multimer analysis. Adhesion of fluo- influence of fractalkine on endothelial function and platelet reactivity were ana- rescence labelled platelets was determined using time-lapse microscopy and com- Methods: Fractalkine levels were determined by ELISA. Platelet surface expres-
Results: All VWF multimers, i.e. from low to high molecular weight, bound to both,
sion of P-selectin and the fractalkine receptor (CX3CR1) were analyzed by flow- collagen I and III at low to high shear rates. Wilate® mediated stable platelet adhe- cytometry. Vascular function of isolated aortic rings was assessed in organ bath sion to collagen at high shear rates applying physiological concentrations of 1 IU/ Results: Fractalkine serum levels were increased in CHF patients (CHF:
Conclusion: The established in vitro flow-chamber model was successfully used
1548±196pg/mL; Control: 968±191pg/mL, p<0.05), and high fractalkine levels for the determination of VWF activity under defined shear rate conditions in real were found in patients with impaired clopidogrel responsiveness measured by the time. Our system represents a promising device for the investigation of the im- P2Y12-specific platelet-reactivity-index (PRI) (PRI>50 %: 1526±98pg/mL; PRI pact of VWF nativity, multimer size and triplet structure on VWF function under <50 %: 744±137pg/mL, p<0.01). In CHF rats following coronary ligation, serum and urine levels of fractalkine were significantly increased compared to control (serum:CHF: 1509±168pg/mL; Control: 1181±58pg/mL, p<0.05). CHF-rats displayedimpaired response to clopidogrel (PRI: CHF64 %; Sham: 30 %, p<0.05). Frac-talkine significantly attenuated endothelial function in CHF rats and augmentedP-selectin expression on platelets from CHF rats. Expression of CX3CR1 on theplatelet surface was increased in CHF rats (CX3CR1 mean fluorescence intensity: Congress Program: 53rd Annual Meeting - Society of Thrombosis and Haemostasis Research Tagungsprogramm: 53. Jahrestagung der Gesellschaft für Thrombose- und Hämostaseforschung e.V. - GTH morbidity and mortality. Thus, we asked, whether YKL-40 is elevated in MO Anti-inflammatory and antinociceptive effects of the direct thrombin inhibitor hirudin
patients and might contribute to the increased CVD event rate in MO.
Design and Methods: 17 patients with MO were included, who were studied before
Lopez M1, Navarro M2, Rosales R1, Avila C1, Nowak G3, Vazquez E1 (BMI:47±6kg/m2) and after weight loss by gastric bypass surgery (BMI:34±6kg/ 1Ivic, Caracas, Venezuela 2Universidad de Carabobo, Venezuela 3University Hospital Jena, Germany m2). Observation period: 17.4±2.2months. Patients were compared to 20 age/ Objectives: Increasing evidence points to extensive cross-talk between inflamma-
sex-matched controls (CO;BMI:22±4kg/m2). We determined fasting Monocyte- tion and coagulation, whereby inflammation leads not only to activation of coagula- chemoattractant-Protein-1 (MCP-1), glucose, insulin and lipids and insulin and glu- tion, but coagulation also considerably affects inflammatory activity.The purpose of cose obtained in a two hour glucose tolerance test, and HOMA-Insulin Resistance.
this study was to investigate the effect of the thrombin inhibitor hirudin on an acute All tests were performed before and after bariatric surgery.
Results: YKL-40 values were tripled (123±61ng/ml) in MO vs. CO. After a weight
Design and Methods: Acute inflammation was produced by subplantar injection of
loss of 40±18kg, YKL-40 levels were reduced to 85±42ng/ml, however, were still 0.1 ml of 2 % carrageenan in a hind paw of male Sprague-Dawley rats. Paw volume doubled vs. CO. Delta YKL-40 (Preoperative minus postoperative) were related was measured before and at different time intervals after carrageenan injection. In to deltas of fasting insulin (R=0.754,p=0.005), HOMA-IR (R=0.789,p=0.002) non-inflamed animals was injected saline instead carrageenan. Hirudin was admin- and MCP-1 (R=0.676,p=0.016). Multivariate regression demonstrated that pre- istered subcutaneously 30 minutes before carrageenan injection. Hotplate latency operativeMCP-1 had the strongest independent association with preopera- test was used to quantify antinociception. Leukocyte count was determined with an tiveYKL-40 (beta=0.805,p=0.01), as had the deltaHOMA-IR with deltaYKL-40 automated counter. C Reactive Protein (CRP) levels were measured with a sand- wich enzyme-immunoassay. Fibrinogen levels were determined by a modification Conclusions: The novelty of this study is the significant elevation of YKL-40 in MO
of the method of Ratnoff and Menzie (1954).
and that these elevated levels can be lowered by weight loss. The significant asso- Results: Injection of carrageenan in the rat paw induced an edema reaching its
ciation with MCP-1 levels is tempting and invites to speculate that MCP-1 attract- maximum after 4 h and decreasing over 96 h. Leukocyte count, CRP and fibrino- ing monocytes to plaque site and YKL-40 being involved in plaque rupture might gen levels reached a maximum 12h, 8h and 36h after carrageenan injection, respec- together be responsible for the increased CVD risk.
tively. Hirudin showed a significant inhibition of carrageenan-induced edema andincreased the latency period in the hotplate test. Leukocytes count and CRP levels were significantly decreased in hirudin treated rats. High fibrinogen levels present Effect of anti-glycemic control on diminished endothelial progenitor cells in type 1
in inflamed rats were not affected by hirudin treatment.
diabetic children
Conclusions: These results suggest that a single application of hirudin prior to car-
Schernthaner G1, Rami B2, Satler M1, Höllerl F1, Höbaus C1, Schober E2, Schernthaner G3 rageenan-induced inflammation has significant anti-inflammatory and antinocicep- Medical University Of Vienna, Internal Medicine II, Vienna, Austria, 2Medical University Of Vienna, Paediatrics And Adolescent Medicine, Vienna, Austria, 3Rudolfstiftung Vienna, Medicine I, Vienna, Severe arteriolar necrosis with renal thrombotic microangiopathy in a septic porcine
Objectives: The risk of cardiovascular death before the age of 40 is 20-fold increased
2-hit model
in patients with type 1 diabetes mellitus (T1DM) compared with nondiabetics. The Bockmeyer C2, Simon T1, Marx G1, Reuken P1, Krusche C2, Lösche W1, Becker J2, Claus R1 mechanisms mediating this risk are not completely understood. Since endothelialprogenitor cells (EPC) predict cardiovascular morbidity and mortality in nondia- 1University Hospital Jena, Germany 2Institute for Pathology, MHH betics, we performed a longitudinal study in T1DM children, enumerated EPC and The balance between VWF and ADAMTS13 is assumed to be involved in the development of sepsis-associated thrombotic microangiopathy (TMA).We hypoth- Design and Methods: 120 children, 90 T1DM and 30 controls (CO) were included
esized that a decreased ADAMTS13-activity in a porcine 2-hit model of hemor- and matched for age, gender and BMI. EPC (CD34+/CD133+/CD309+) were enu- rhage and sepsis is associated with laboratory and morphological findings of TMA.
merated by flow cytometry at the beginning and one year thereafter, when the Animals (n=21, BW 24 kg) were subjected to hemorrhagic shock (45 min). After patients were scheduled for their routine follow up examination. In order to anal- re-transfusion, sepsis was induced via intraperitoneal implantation of fibrin clot to yse changes of variables during the one year observation time, delta values were restrain continuous release of 109 vital E.coli. Blood samples for determination of ADAMTS13-activity, VWF:Ag and platelet count, creatinine and hematocrit for Results: EPC were reduced in T1DM children vs. CO: 607±368vs.1037±504,p<0.001.
normalization were drawn. Directly postmortem we examined renal TMA develop- Multivariate regression revealed that HbA1c was the strongest independent predic- ment (CD61 staining) and ischemic acute tubular necrosis (epithelial brush border tor of EPC (Beta=-0.319,p=0.003). Overall glycemic control at the beginning and staining).ADAMTS13 activity remained stable after hemorrhage,however,declined end of study did not differ (7.8±1.2vs.7.8±1.2rel.%,p=ns). However, we observed stepwise during sepsis accompanied by a strong drop off in platelet count (356 to HbA1c changes during the study in the individual patient of -4.30/+3.10 rel. %.
142 GptL-1). Moderate increase in VWF:Ag was found. Development of renal dys- Calculating the mean change of EPC per mean change of HbA1, resulted in an function was indicated by a slight, but significant increase in plasma creatinine con- increase of 93 EPC per each 0.1rel.% reduction of HbA1c and vice versa.
centration (107.0 vs. 141.0 µmolL-1, p<0.05). Characteristic findings for TMA with Conclusions: In conclusion, this is the first study demonstrating diminished EPC in
glomerular microthrombi in kidneys, fibrinoid necrosis of preglomerular arterioles children with T1DM. The observed increase of EPC in children with improved dia- and interlobular arteries with interstitial bleeding were observed. Glomerular lumi- betic control within one year suggest that optimization of glycemic control could be nal platelet activation and ischemic acute tubular necrosis were most prominent in relevant in reducing the high cardiovascular disease burden in T1DM patients.
septic animals in comparison to sham-operated ones. Similar to patients with severesepsis a decline in ADAMTS13 activity in association with platelet consumption was observed in a septic porcine model whereas renal TMA was present in one ani- Factor Seven Activating Protease: a link between inflammation and coagulation in
mal. Therefore, we present an animal model to perform functional studies focusing coronary artery disease
on pathophysiological mechanisms of sepsis-associated TMA.
Parahuleva M1, Wolter J1, Maj R1, Staubitz A1,3, Hölschermann H2, Tillmanns H1, Erdogan A1, Kanse S31Department of Internal Medicine, Division of Cardiology and Angiology, University Hospital of Giessen PP5.2 Atherosclerosis
and Marburg, Location Giessen, Germany, 2Department of Internal Medicine, Division of Cardiologyand Angiology, Hochtaunus Kliniken gGmbH, Bad Homburg v.d. Höhe, Germany, 3Institute fürBiochemie, Justus-Liebig-Universität, Giessen, Germany Introduction and hypothesis: FSAP may be involved in the progression of athero-
YKL-40, a player in extracellular matrix remodeling, is increased in morbid obesity
sclerosis and the development of associated clinical events. It is present in unstable but declines with weight loss
lesions and its plasma level and activity are increased in patients with coronary Hempen M1, Kopp H2, Elhenicky M1, Höbaus C1, Brix J2, Koppensteiner R1, Schernthaner G2, artery disease (CAD). The molecular mechanism, however, by which circulat- ing FSAP influences the progression of CAD is not yet entirely understood. The 1Medical University Of Vienna, Department Of Internal Medicine II, Vienna, Austria, 2Rudolfstiftung present study was performed to examine the relation between FSAP and the pro- Hospital, Department Of Medicine I, Vienna, Austria inflammatory activation of macrophages.
Methods: The influence of FSAP on the activation of transcription factors of the
Objectives: YKL-40 is a protein involved in extracellular matrix remodeling. Ele-
NFkappaB family and cAMP response element-binding protein (CREB) was vated YKL-40 was found in advanced stages of atherosclerosis/coronary heart dis- assessed by electrophoretic mobility shift assays. Degradation and phosphorylation ease. Morbid Obesity (MO) is associated with high cardiovascular disease (CVD) of the inhibitor protein IkBα, ICAM-1, IL-6, and TF mRNA were analysed.
Congress Program: 53rd Annual Meeting - Society of Thrombosis and Haemostasis Research Tagungsprogramm: 53. Jahrestagung der Gesellschaft für Thrombose- und Hämostaseforschung e.V. - GTH Results: FSAP treatment (20 µg/ml) induces IkappaB-dependent NF-kappaB
PP5.3 Cellular Activation and Signaling
activation in freshly isolated human monocytes in a time-dependent fashion. Itinduces the phosphorylation and proteolytic degradation of the inhibitor proteinIkappaBα. The phosphorylation of p65 was induced by FSAP, which is known to contribute to the enhancement of DNA-binding activity of NF-kappaB. In parallel, Inhibition of platelet alpha-granule release strongly attenuates granulocyte adhesion
FSAP induced the expression of ICAM, IL-6, and TF, genes known to be under the and activation on decellularized porcine heart valve tissue
control of NF-kappaB. Consistent with this, aprotinin, a pharmacological inhibitorof FSAP, blocks FSAP-induced gene expression. In contrast, CREB phosphoryla- Stelzmueller M1, Kasimir M1, Bastian F1, Nigisch A1, Simon P2, Weigel G1 tion and activation was not detected in FSAP-treated monocytes.
1Medical University Vienna/ Department Of Cardiothoracic Surgery, Vienna, Austria 2Saad-Specialists Conclusions: Biological functions of FSAP in macrophages extend beyond its role
in promoting thrombosis/fibrinolysis. Thus, FSAP may play an important role in Objectives: This in-vitro study was designed to investigate the impact of platelet
atherosclerosis by enhancing the inflammatory response of human macrophages as adhesion on attraction and activation of polymorphonuclear leukocytes (PMN) by decellularized xenogeneic tissue.
Methods: Cryostat sections of decellularized porcine heart valves were sequentially
incubated with platelet-rich plasma (PRP) and isolated, autologous PMN. In fur- Vascular (coronary/cerebral) events following retinal vein occlusion: A 8-yr follow
ther experiments, platelets were pre-incubated with either Cytochalasin D (CytD) or iso-butyl-methyl-xanthine (IBMX) to block platelet alpha-granule release. To Guida A, Di Capua M, Coppola A, Macarone Palmieri N, Tufano A, Di Minno M, Cimino E, Di Minno G investigate the involvement of the complement system, specimens were exposed to PRP that had been pre-incubated with 10 mm EDTA. At the end of the incuba-tions, sections were washed and fluorescently stained for CD 41, thrombospondin-1 We have followed-up for 8 yrs (mean, 7.9”b2.7 yrs) 117 consecutive patients (61 (TSP-1), CD45, CD11b, and the complement factor iC3b.
M, 56 F; mean age 54±13; age at the event 51±13) referred to our Centre for fluo- Results: Laser scanning microscopic examination revealed the binding of multiple
rangiographically documented retinal vein occlusion (RVO; 62 central; 48 branch, activated platelets to the decellularized porcine tissue. Platelet adhesion was asso- BRVO; 7 both types in different eyes). As many as 202 matched apparently healthy ciated with upregulated expression and secretion of TSP-1. Pre-treatment of tissue individuals (105 M, 97 F; mean age 52±12) from the same geographic area served as specimens with PRP induced a strongly enhanced binding and activation of sub- controls. At the time of the RVO, hypertension (64.6 % vs. 28.4 %; OR 4.6 CI 2.8– sequently added PMN. Blockade of platelet alpha-granule release by either CytD 7.5; p<0,0001) and diabetes mellitus (17.8 % vs. 8.1 %; OR 2.5 CI 1.1–5.5; p<0.05) or IBMX markedly reduced the deposition of TSP-1 and significantly decreased had a higher prevalence in patients than in controls. No other cardiovascular risk the adhesion of PMN. Although inhibition of complement activation by addition factor nor inherited/acquired thrombophilic abnormalities significantly differed in of EDTA to the PRP preparations inhibited iC3b deposition it failed to prevent controls and patients. In contrast, BRVO differed from CRVO as to age and preva- lence of diabetes mellitus, overweight and hypertension (55 vs. 47 yrs; p<0.0022; Conclusion: Platelet adhesion to acellular porcine heart valve tissue induces TSP-1
9.7 % vs. 8.7 %; 83.9 % vs. 57.8 %; 78.7 % vs. 55.9 % respectively; p always <0.05).
secretion with subsequent PMN binding and expression of the activation marker Eight yrs after the RVO, 90/117 patients (77 %) gave their consent to undergo a CD11b. The interaction of platelets with the decellularized matrix seems therefore follow-up visit. Fifty-eight of them (64 %) had experienced new vascular events: to play a key-role in the early non-specific inflammatory response toward acellular coronary/cerebral non-fatal ischemic events (n=38); retinal events (n=10), venous xenogeneic implants independent from complement activation.
thrombosis (n=10). In 22/90 patients (24 %) in whom antiplatelet agents wereemployed throughout the follow-up, a lower prevalence of overall vascular recur- rence (45.4 %[10/22] vs. 70.6 %[48/68], p=0.06) was found. These data are consistent The extracellular adherence protein (Eap) of staphylococcus aureus activates
with RVO as being an atherosclerosis-related event, and support the possibility thatthe correction of hypertension and/or diabetes mellitus is a major direction to be platelets via the ATP-gated P2X1cation channel and Fcgamma RIIA
Niemann S1, Hussain M2, Pohl S1, Schifferdecker T1, Schulte A1, Bertling A1, Heilmann C2,Kehrel B1 1Anästhesiologie und operative Intensivmedizin, Exp. und Klin. Hämostaseologie, UKM, Münster, Cholesterol-induced apoptosis is associated with downregulation of the activated
Germany 22Institut für Medizinische Mikrobiologie, Universitätsklinikum Münster, Münster, leukocyte cell adhesion molecule (ALCAM) in human monocytic cells
Rauch S1, Rosenkranz A1, Meyer-Kirchrath J1, Schrör K1, Rauch B1 Objectives: The extracellular adhesion protein (Eap), secreted by the major patho-
1Institut für Pharmakologie und Klinische Pharmakologie, Düsseldorf, Germany gen Staphylococcus aureus, is known to influence human immunity. The knowndirect interaction with fibrinogen and some integrins prompted us to study the Migration of monocytes plays an important role in development and progression of atherosclerosis. The activated leukocyte cell adhesion molecule (ALCAM/CD166) Methods: Platelet activation was studied by aggregometry and flow cytometry.
is important for cell migration and leukocyte invasion. The present study investi- Results: Eap (1–10 µg/ml) activated human platelets in a dose, time, and temper-
gates the impact of cholesterol-enrichment on the expression of ALCAM in the ature dependent manner. It induced fibrinogen and surprisingly von Willebrand human monocytic cell line U937. Monocytic U937 cells were enriched with choles- factor binding as well as platelet aggregation. Activated platelets bound thrombos- terol by incubation with methyl-beta-cyclodextrin (MbCD)-cholesterol-complex.
pondin-1 as well as the coagulation factors VIIa, VIII and XIII and annexin V. Eap Expression of adhesion molecules was determined by flow cytometry; apoptosis induced the secretion of the granules and the formation of platelet-leukocyte asso- by Annexin-V FITC/PI (propidium iodide) double-label cytometry. Migration of ciates. Eap treatment lowered the number of free SH-groups on the platelet surface calcein-AM-loaded U937 cells was quantified in 3 µm-chemotaxis chambers by significantly and Eap action on platelets was blocked by SH-reactive DTNB, PAO, fluorescence of transmigrated cells. Incubation of monocytes cells with cholesterol pCMBS, gliotoxin and the protein disulfate isomerase inhibitor bacitracin. Fab frag- (10 - 100 µg/mL) for 18 h induced a concentration-dependent increase in early ments of the Fcgamma RIIa inhibitory antibody IV.3 (2µg/ml) blocked Eap action and late phase apoptosis, while MbCD alone had no effect. Increased apoptosis on platelets and the known inhibitor of Fcgamma RIIa signal transduction, the rate was associated with a reduction of ALCAM expression by >50 %. In contrast, tyrosine kinase inhibitor Piceatannol, inhibited as well. NF449, which selectively expression of VCAM-1 (vascular cell adhesion molecule-1) was strongly increased blocks P2X1 receptors, blocked platelet activation by Eap.
and ICAM-1 (intercellular cell adhesion molecule-1) levels were not affected by Conclusions: The extracellular adherence protein (Eap) of Staphylococcus aureus
cholesterol loading. Pretreatment with the nonselective caspase/apoptosis inhibi- activates platelets via the ATP gated cation channel P2X1 and Fcgamma RIIA tor Q-VD-OPh (100 µmol/L) partially prevented cholesterol-induced alteration (CD32). P2X1 cation channel and Fcgamma RIIa are sensitive to SH-Blockers.This of adhesion molecule expression. Cholesterol loading was also associated with an might explain why Eap induced cell activation was blocked by thiol reactive tools.
80 % reduction of cell migration towards 10 % serum. Q-VD-OPh partially res- Whether the found signalling way for Eap is general for other blood and vascular cued migration capacity in cholesterol-rich monocytes. This effect was prevented by addition of ALCAM-neutralizing antibodies (10 µg/mL), but not an isotype-matched IgG. Cholesterol-induced apoptosis in monocytic cells is accompanied byreduced expression of ALCAM. Loss of ALCAM under conditions of cholesterol-loading and apoptosis attenuates monocyte migration, a mechanism which mayexpedite vascular lesion formation.
Congress Program: 53rd Annual Meeting - Society of Thrombosis and Haemostasis Research Tagungsprogramm: 53. Jahrestagung der Gesellschaft für Thrombose- und Hämostaseforschung e.V. - GTH (mainly platelets) activation, using a double centrifugation at Room Temperature.
CD11B-positive microparticles are an independent predictor of cardiovascular events
This plasma is then diluted and introduced into a microwell coated with a specific in patients with coronary artery disease
Anti-TF MoAb. Microparticles exposing TF are captured, and their activity is mea-sured through Factor Xa generation. The revelation mixture contains Factor VII Faille D123, Frère C123, Cuisset T4, Quilici J4, Morange P123, Juhan-Vague I123, Bonnet J4, Alessi M123 or (better) VIIa in a constant and in excess concentration, Factor X and calcium.
1INSERM, UMR 626, Marseille, France, 2Aix-Marseille Université, Faculté de Médecine, Marseille, There is a direct dose response relationship between MP-TF concentration and France, 3Laboratory of Hematology, CHU Timone, Marseille, France, 4Department of Cardiology, Factor Xa generated. This Factor Xa is then measured by its activity on a specific chromogenic substrate and colour development is measured at 405 nm. The assay is Many cells of the vascular compartment play a key role in the pathogenesis of coro- calibrated with recombinant TF, at a known concentration, which is relipidated at a nary artery disease (CAD) and its complications. Accurate determination of the well defined Phospholipids concentration (12.5 % PS) using liposomes, and spiked activation status of these various cell types might thus be helpful to identify coro- into normal plasma. The assay has a dynamic range from 20 pg/ml to 1,000 pg/ml TF, nary patients at high risk for future cardiovascular events (CVE). Microparticles and is performed within less than 2 hours. Tested plasma can be used diluted 1:2 or (MP) are membrane fragments known to be reliable markers of cell apoptosis and at higher dilutions according to the expected MP-TF concentration in the assayed specimen. This assay introduces a new analytical tool for measuring an emerging Objectives: To determine if MP levels from different cell types were associated with
marker of blood activation in circulatory or malignant diseases. The diagnostic and the severity of atherosclerotic lesions and the occurrence of future CVE in patients prognostic value of this marker is amplified by the nature of MP-TF, which are an indicator, but also a trigger for thrombotic pathologies. It offers promising perspec- Design and Methods: Baseline plasma MP levels were measured by flow cytometry
tives for the early diagnosis of thrombotic or malignant diseases.
in 172 patients undergoing coronary angiography at the Timone Hospital (Mar-seilles, France). 77 patients underwent stent implantation and were followed-up for1 month.
PP5.4 Endothelial Cells, Extracellular Matrix and Angiogenesis
Results: 12 CVE were recorded during the follow-up period. In the entire cohort
of patients, levels of leukocyte-derived MP (CD11b+MP) were found strongly but
negatively related to the number of occluded coronary arteries (P = 0.005) and weresignificantly higher in patients under statin therapy (P = 0.001). In the followed-up Regulation of mouse embryonic stem cell commitment into the endothelial lineage by
population, CD11b+MP were lower in patients with CVE (P = 0.0001). This dif- p38 mitogen-activated protein kinase
ference remained significant after adjustment on the number of occluded arteries Binetruy B1, Barruet E1, Peiretti F1, Hadadeh O1, Juhan-Vague I1, Alessi M1 and on the statin therapy status (P = 0.004). Interestingly, monocyte-derived MP 1Inserm U626, Faculte De Medecine, Marseille, France (CD14+MP) were more elevated in patients with future CVE (P = 0.0005).
Conclusions: Leukocyte-derived MP provide a useful tool in identifying patients at
Embryonic stem (ES) cells are able to give rise, in vivo, to all of the three germ high risk of recurrence independently of the severity of atherosclerotic lesions.
layers and, in vitro, to differentiate into a broad variety of cell lineages which giveus large perspectives in regenerative medicine. This process is strictly controlled by the potent morphogen retinoic acid (RA), when added from day 3 to day 5, itinduces ES cell differentiation into neurons, and, conversely, inhibits cardiomyo- Phospholipids in misguided thrombus resolution after splenectomy
genesis, skeletal myogenesis and endothelial cell lineage. We found that p38MAPK Renner M1, Redwan B1, Panzenboeck H1, Jakowitsch J1, Sadushi R1, Kellermair J1, Bonderman D1, activity peaked spontaneously between day 3 and day 5, during mouse ES cell dif- ferentiation and that RA completely inhibited this peak of activity. At the opposite 1Innere Medizin II, Kardiologie, AKH, Wien, Austria of wild type cells, p38α-/- ES cells differentiated spontaneously with or without RAtreatment into neurons, and did not form cardiomyocytes, myocytes or endothelial Purpose: Splenectomy is associated with an increased risk of chronic thromboem-
cells. Similar results were obtained by treating wt ES cells with a p38MAPK-specific bolic pulmonary hypertension (CTEPH). CTEPH is a life-threatening condition chemical inhibitor (PD169316). By genetic and biochemical approaches, we dem- characterized by single or recurrent pulmonary thromboemboli that obstruct or onstrate that the control of p38MAPK activity constitutes an early switch, commit- obliterate the pulmonary vascular bed. The aim of our study was to investigate the ting ES cell into either neurogenesis (p38 off) or several mesodermal lineages (p38 role of phospholipids in the pathogenesis of altered thrombus resolution after sple- on). Furthermore, our results suggest that Flk-1+ cells could constitute the common mesodermal progenitors targeted by p38MAPK in this process. Finally, after stable Methods: We utilized a mouse model of stagnant flow venous thrombosis to char-
transfection of an expression vector, we were able to express an activated form of acterize venous thrombus resolution. Vena cava ligation was performed one month p38α in wtES cells as well as in p38α -/- ES.Analysis of the differentiation capacities after splenectomy. At days 3,7,14 and 28 after vena cava ligation thrombi were harvested for histology and electrospray ionization - mass spectrometry analysis.
Blood samples were collected for FACS.
Results: Thrombus areas of splenectomized mice were significantly larger than
those of controls at all time points (ANOVA, n=8, p<0.03). The composition of
Endothelial progenitor cells, insulin resistance, inflammation, and their associations
phospholipids enclosed in the thrombus was significantly different between thrombi in morbidly obese patients undergoing massive weight loss induced by bariatric
of splenectomized mice and thrombi of control mice. In parallel, whole blood FACS revealed higher counts of CD41-platelet microparticles (day 14: 3216 versus 927 Schernthaner G1, Satler M1, Kopp H2, Kriwanek S2, Koppensteiner R1, Schernthaner G2 cells/µl, p< 0.05) and leukocyte/platelet aggregates (day 14: CD11b/CD41, 56.4 ver- 1Medical University Vienna, Internal Medicine II, Angiology, Vienna, Austria 2Rudolfstiftung Hospital, Conclusion: We suggest that an altered phospholipid profile in thrombus may
derive from platelets and leukocytes after splenectomy. The loss of mechanical fil-
Objectives: Morbid Obesity (MO) is associated with high cardiovascular disease
tering function of the spleen permitting the accumulation of phospholipids in the (CVD) morbidity and mortality. Traditional risk factors only partly explain why life peripheral circulation is a key mechanism of thrombus persistence.
is diminished by 12 years. Since endothelial progenitor cells (EPC) predict CVDevents and death, low EPC in MO patients may contribute to the increased mortal- ity. Thus, we investigated EPC in MO patients before and after weight loss.
Design and Methods: 109 patients with MO (BMI:46±7kg/m2) were compared
Measurement of procoagulant potential of blood microparticles carrying Tissue
with 64 controls (CO) (BMI:23±2kg/m2) and 95 patients with massive weight loss 1.6±0.4 years after bariatric surgery (BMI:33±6kg/m2). Circulating progenitor Laroche M1, Peyrafitte M1, Vissac A1, Amiral J1 cells (CPC,CD34+/133+), EPC (CD34+/133+/309+) and activated EPC (actEPC, 1Hyphen Biomed Research, Neuville sur Oise, France CD34+/133+/309+/31+) were enumerated by FACS. EPC/CPC ratio, actEPC/EPC Blood microparticles (MPs) have high diagnostic and prognostic interest, not only ratio and concentrations of fasting MCP-1, IL-18, hsCRP were determined.
for circulatory diseases, but also for inflammatory, malignant or infectious patholo- Results: CPC, EPC and actEPC were drastically reduced in MO patients compared
gies. These MPs objectivate blood cell activation, but they also contribute to the to CO. CPC, EPC and actEPC were significantly higher after a mean weight loss of disease course worsening through their procoagulant effect. A special focus con- 39±19 kg, but did not reach the numbers of CO. Reduction of EPC and actEPC were cerns MPs which expose Tissue Factor (TF), involved in thrombotic diseases. From significantly associated with BMI, insulin resistance (fasting-insulin/glucose,2-hour- the studies of JM Freyssinet‘s group, we adapted a new method which specifically insulin/glucose,HOMA-IR), and markers of inflammation (hsCRP,MCP-1,IL-18).
measures the procoagulant activity of blood MPs carrying Tissue Factor (MP-TF).
Conclusions: The novel finding of this study is a significant impairment of EPC in
Plasma is obtained with the specific cautions required for avoiding ex-vivo cell MO patients and that high levels of adipocyte-derived IL-18 suppress activation of Congress Program: 53rd Annual Meeting - Society of Thrombosis and Haemostasis Research Tagungsprogramm: 53. Jahrestagung der Gesellschaft für Thrombose- und Hämostaseforschung e.V. - GTH EPC. Marked weight loss by bariatric surgery resulted in an increase of CPC, EPC, their activities are changed by ectodomain cleavage and release as soluble forms actEPC and respective ratios. It is tempting to speculate that the reduced CVD (sEPCR and sTM). In the current study we analysed the mechanisms involved in mortality of MO patients after weight loss could partly be explained by increase of the regulation of EPCR and TM shedding.
EPC/act-EPC, both assumed to have a critical role in vascular repair mechanism.
Methods: The activations of ERK-1/2, p38 MAPK, and c-Jun N-terminal kinase
(JNK) were studied by cell-based ELISA and the levels of sEPCR and sTM by
The gut microbiota triggers Tissue Factor-dependent angiogenesis
Results: IL-1 and TNF-α, but not IFN-γ or IL-6 induced a rapid sEPCR release in
human umbilical vein endothelial cells (HUVEC) correlating with the activation of
p38 MAPK and JNK. The cleavage of EPCR was also up-regulated by thrombin, 1Göteborg University, Department Of Clinical Bacteriology, Göteborg, Sweden 2Göteborg University, anisomycin, a JNK agonist, PMA, calcium-ionophore, thiol alkylators and oxidants, The Wallenberg Laboratory for Cardiovascular Research, Sweden as well as by lipid raft disruptors. Both basal and induced shedding was prevented Objectives: Germ-free (GF) mice exhibit arrested formation of capillaries in the
by metalloproteinase inhibitor, TAPI-0, and by radical scavenger, N-acetylcysteine small intestinal villi compared with ex-GF mice that were colonized with normal (NAC). However, the intracellular generation of radical oxidant species did not gut microbes (CONV-D) or conventional-raised (CONV-R) animals. The mecha- correlate with the EPCR release and antioxidants failed to block the EPCR shed- nisms underlying these phenotypic differences are unknown. In the present study ding. Further results suggest that a direct targeting of thiol groups involved in the we identify angiogenic factors responsible for microbially-induced capillary net- control of metalloproteinase activities is responsible for the observed effects of work formation and assess whether microvascular angiogenesis in the gut is depen- NAC on EPCR shedding. Similar to EPCR the release of TM in HUVECs is regu- dent on the signaling function of Tissue Factor (TF).
lated by TAPI-0 sensitive mechanisms, but NAC profoundly increased the cleavage Design and Methods: To elucidate the potential role of TF, ex-GF mice were
injected intraperitoneally with an anti-TF antibody or an isotype control antibody Conclusions: The shedding of EPCR and TM is strongly regulated by different cell
prior to a 14 day colonization with a cecal microbiota harvested from CONV-R signaling pathways and thiol-oxidizing or reducing agents.
mice that stimulates capillary network formation. The vascular marker CD31(PECAM-1) was used to quantify villus vascularization by immunohistochemistry and qRT-PCR. To uncover proangiogenic genes upregulated in response to micro- Detecting heparanase inhibitors with a novel heparanase activity assay fit for
bial colonization qRT-PCR-analyses were performed on small intestinal tissues col- lected from GF, CONV-R and CONV-D mice that were treated with either anti-TF Results: We found that microbial colonization of the intestine leads to formation of
Pharmazeutisches Institut, CAU Kiel, Germany an intricate vascular network. This process was efficiently blocked by administra- Human heparanase overexpression promotes tumour metastasis, angiogenesis tion of anti-TF antibody prior to microbial colonization of the animals. A distinct and is associated with poor prognosis. Therefore, heparanase inhibitors (HI) are set of proangiogenic genes was identified to be upregulated in a TF-dependent considered as promising candidates for tumour therapy. So far, no simple, rapid manner following microbial colonization.
assay is available for testing potential HI. The aim of this study was to develop an Conclusion: Our results suggest that the signaling function of TF is crucial for
assay based on the finding that fondaparinux represents a substrate for heparanase microbiota-induced formation of capillary networks in small intestinal villi. This and on the idea to quantify the degradation of fondaparinux by means of its anti- mechanism is potentially relevant in inflammatory bowel disease (IBD), where factor Xa (aXa)-activity. Structurally defined semi-synthetic glucan sulfates (GS) were examined for their heparanase inhibitory activity. The assay was establishedas a two step micro-plate-assay. First, heparanase is incubated with the substrate fondaparinux and different concentrations of potential HI. After stopping the reac- Acute pro-coagulatory response of the endothelium to melanoma cells
tion by freezing, the remaining substrate is quantified by its aXa-activity in a chro-mogenic substrate assay (bovine FXa, antithrombin, S2222). Initially, the following Strozyk E1, Kerk N1, Schnaeker E1, Pöppelmann B1, Huck V1, Gorzelanny C1, Schneider S1 assay parameters were evaluated and optimized: incubation time, concentrations 1Klinik u. Poliklinik für Hautkrankheiten, Münster, Germany of substrate and heparanase, heparanase stability and inactivation method, as well Objective: Tumor cell spreading is accompanied by an intrinsic pro-coagulatory
as aXa-assay conditions. The GS structure-dependently inhibited heparanase with activity and by inflammatory conditions. Endothelial von Willebrand factor (VWF) IC50 values ranging between 0.5ng/mL and 5000ng/mL. Their activity increased is emerging as a key protein in both these processes.
with increasing degree of sulfation (DS) and decreasing molecular weight (MW).
Methods: Supernatants (SN) obtained from invasive melanoma cell lines MV3 or
Beta-1,3-GS were about 10 times more active than alpha-1,4/1,6-GS with similar WM9 were tested for human umbilical vein endothelial cells (EC) activation either DS and MW. In conclusion, a novel chromogenic heparanase activity micro-plate- by VWF release using ELISA and perfusion experiments or NFkappaB activation assay suitable for high-throughput screening has been developed. By studies on analysed by EMSA. Expression of NFkappaB-dependent genes was assayed by structure-activity relationships using this assay, beta-1,3-GS were identified as PCR and protein content by ELISA or FACS. Melanoma-mediated thrombin gen- potent HI, which may contribute to their proven antimetastatic activity.
eration and activity was measured using chromogenic substrate S-2238.
Results: MV3 cells induce an instantaneous, massive release and immobiliza-
tion of ultra-large VWF (ULVWF) at the luminal endothelial membrane via EC Aortic valve stenosis progression parallels changes in extracellular matrix gene
PAR-1 activation initiating tumor cell adhesion. WM9 cells, in contrast, activate the expression and angiogenesis
NFkappaB pathway in EC followed by an up-regulation of IL-6 and TF expression.
Panzenböck A1, Martischnig A1, Bonderman D1, Jakowitsch J1, Seitelberger R2, Rosenheck R1, Moreover, both melanoma cell lines express TF which contributes to generation ofthrombin in blood plasma.
Conclusion: Melanoma cells can directly (via secreted mmPs) or indirectly (via TF-
1Universitätsklinik für Innere Medizin II, Wien, Austria 2Universitätsklinik für Chirurgie mediated thrombin generation) activate PAR-1 on EC leading to an acute luminal Background: Aortic valve disease is the most frequent native valve disease in
ULVWF release. Binding of melanoma cells to ULVWF supports adhesion and Europe, and the third most frequent cause of cardiovascular death. The biological facilitates reciprocal communication such as activation of the NFkappaB pathway.
mechanisms underlying aortic valve degeneration, ultimately resulting in calcific Thus, ULVWF release and stabilisation may be enhanced by up-regulation of TF aortic stenosis show similarities to atherosclerosis. We investigated extracellular or production of IL-6 which diminishes the activity of VWF-degrading protease matrix (ECM) molecules and angiogenesis as important components of atheroscle- ADAMTS13. We hypothesize that the switch from an anti- to a pro-inflammatory and pro-coagulatory surface of EC plays a pivotal role in melanoma cell extravasa- Methods: Aortic leaflets from valve replacement surgeries and heart transplants
were collected in the operating room after a careful echocardiographic valida-tion of mean valve gradients (mvg) within 7 days of surgery. RNA expression profiles of healthy (control), as well as sclerotic (mvg=6±2mmHg, mean±SD), Regulation of endothelial protein C receptor and thrombomodulin shedding in human
mildly (mvg=22±12mmHg), moderately (mvg=49±16mmHg) and severely stenotic endothelial cells
(mvg=63±15mmHg) aortic valve leaflets (n=5 per group) were analyzed using anAffymetrix Human Gene 1.0 ST Array. Genes with an absolute fold change of ±2.0 Siegert G1, Menschikowski M1, Hagelgans A1 in at least 4 samples per group were selected for further analysis.
1Institute Of Clinical Chemistry And Laboratory Medicine, Dresden, Germany Results: Collagen types I, III, V, XIV, XV and XXI as well as cathepsin S, B, D
Objectives: Endothelial protein C receptor (EPCR) and thrombomodulin (TM)
and K and perlecan showed a valve gradient-dependent increase of expression.
play pivotal roles in coagulation, cell proliferation, and inflammation. However, Expressions of collagens type Ia1, Ia2, IIIa1 and Va1 and cathepsins s and D Congress Program: 53rd Annual Meeting - Society of Thrombosis and Haemostasis Research Tagungsprogramm: 53. Jahrestagung der Gesellschaft für Thrombose- und Hämostaseforschung e.V. - GTH showed a significant correlation with stenosis progression (14,5 (9–17) m/sec/year, median(range), p<0,05). The antiangiogenic ECM molecule chondromodulin was ACE inhibition reduces monocyte MFG-E8 and MCP-1 expression associated with
at a low level across the spectrum of mvgs, while VEGF decreased in parallel to increased serum fractalkine levels in rats with chronic heart failure
increasing mvg.
Conclusion: The data demonstrate an increase in ECM and suppression of angio-
Pfrang J1, Fraccarollo D1, Ertl G1, Bauersachs J1, Schäfer A1 genesis in direct correlation with mvg and aortic stenosis progression.
1Medizinische Universitätsklinik Würzburg, Germany
Introduction: Chronic heart failure (CHF) still remains a cause of high morbidity
and mortality. Immune activation and inflammation influence CHF pathogenesis.
Correlation of different circulating endothelial progenitor cells to stages of diabetic
The pro-inflammatory chemokine fractalkine induces monocyte activation. Frac- retinopathy – first in vivo data
talkine promotes mfg-e8 expression on macrophages. We investigated whether (a)Fractalkine serum levels are significantly enhanced in CHF, (b) high fractalkine Hoellerl F1, Brunner S2, Satler M1, Elhenicky M1, Binder S2, Schernthaner G3, Schernthaner G1 levels influence mfg-e8 and MCP-1 expression on peripheral blood monocytes, and 1Medizinische Universität Wien, Innere Medizin II, Angiologie, Wien 2Krankenanstalt Rudolfstiftung, (c) these proinflammatory markers can be positively modulated by a standard CHF Wien, Ophthalmologie, Wien, 3Krankenanstalt Rudolfstiftung, Medizin I; Wien, Österreich Objectives: To investigate vasculogenic circulating progenitor cells (CPC), endothe-
Methods: In rats with CHF following chronic coronary ligation for 10 weeks, frac-
lial progenitor cells (EPC), and activated EPC in patients with type 1 diabetes mel- talkine levels were determined in serum and urine using ELISA. Peripheral blood litus (T1DM) with or without diabetic retinopathy (DR).
monocytes were isolated via Histopaque gradient. Mfg-e8 and MCP-1 protein Design and Methods: A case-control study comparing 90 patients with T1DM with
expression on these monocytes were assessed by Western Blotting and expressed as and without DR was performed. Patients were studied and staged for retinopathy mean±S.E.M. Analysis was done via unpaired Student’s t-test. P values<0.05 were according to the Early Treatment of Diabetic Retinopathy Study (ETDRS) clas- considered statistically significant.
sification. 90 patients were included: 30 without DR (CO), 30 with mild non-prolif- Results: Fractalkine serum and urine levels were significantly higher in CHF com-
erative DR (mNPDR), 10 with moderate-severe NPDR (msNPDR), 10 with mild- pared to sham-operated rats (Serum: CHF: 1509±168pg/mL; Sham: 1181±58pg/mL, moderate proliferative diabetic retinopathy (mmPDR) and 10 with high-risk PDR p<0.05). Mfg-e8 and MCP-1 expression on peripheral blood monocytes were sig- (hrPDR). CPC (CD34/CD133), EPC (CD34/CD133/CD309) and mature actEPC nificantly enhanced in CHF animals (mfg-e8: CHF: 1,36±0,36 abitrary units (au); (CD34/CD133/CD309/CD31) were enumerated by flow cytometry.
Sham: 0,79±0,08au; MCP-1: CHF: 1,79±0,29au; Sham: 1,01±0,17au, p<0.05 ) and Results: EPC were reduced in mNPDR (114±66; p<0.001) and msNPDR (77±40;
p=0.042) vs CO (244±115). In contrast, EPC were unchanged in mmPDR (248±155) Conclusion: Fractalkine serum levels and monocyte expression of its downstream
vs CO. Strikingly, EPC were augmented in hrPDR (389±124) vs all other stages.
target mfg-e8 were significantly increased in experimental CHF as was the burden Numbers of undifferentiated progenitor cells (CPC) did not differ between of MCP-1. Ramipril reversed the pro-inflammatory changes in CHF monocytes, CO,mmPDR and hrPDR.A three times augmentation of actmature EPC in hrPDR indicating that ACE inhibition might beneficially modulate chemotactic/athero- (325±118; p<0.001) vs CO (100±49), but also against all other stages of DR was observed. The percentage of actmature EPC/EPC was augmented in an ETDRSclassification dependent way.
Conclusions: In type 1 diabetic patients with diabetic retinopathy, circulating pro-
Bivalirudin reduces platelet and monocyte activation after elective percutaneous
genitor cells (CPC) show a stage-related regulation. In non-proliferative retinopa- coronary intervention
thy a reduction of CPC, in proliferative retinopathy an dramatic increase of matu-reactivated endothelial progenitor cells was observed.
Demetz G1, Steppich B1, Sibbing D1, Stein A1, Schömig A1, Kastrati A1, von Beckerath N1, Ott I11Deutsches Herzzentrum TU München, GermanyConcomitant antithrombotic therapy is essential in prevention of ischemic events PP5.5 Inflammation, Cytokines and Auto-Immunity
in percutaneous coronary intervention (PCI) and stenting. With new anticoagulantmedications being developed and applied in PCI, this raises the question of possibleinteractions with platelet and leukocyte activation. We, therefore, sought to investi- gate the influence of bivalirudin and heparin in platelet and leukocyte activation in A role for Flk-1 and PECAM-1 in monocyte recruitment into resolving thrombi
Redwan B1, Renner M1, Kellermaier J1, Panzenböck H1, Jakowitsch J1, Wagner E2, Bonderman D1, Methods and Results: 46 Patients were recruited consecutively in the setting of
the Intracoronary Stenting and Antithrombotic Regimen-Rapid Early Action for
Coronary Treatment (ISAR-REACT)-3 trial and randomly assigned to receive 1Medical University of Vienna, Division of Cardiology, Vienna, Austria 2Institute of Molecular Pathology, either unfactionated heparin or bivalirudin during elective PCI. Surface expression of CD62P (P-Selectin), CD42b (GPIb ), CD40L, PAC-1 on circulating platelets and Background: Angiogenesis and leukocyte recruitment are key components of
CD11b, CD14 and CD15 on circulating leukocytes were evaluated by flow cytom- thrombus resolution. Platelet endothelial cell adhesion molecule-1 (PECAM-1 etry. Cytokine levels of IL-12p70, Tumor necrosis factor (TNF), IL-8, IL-6, IL-1 or CD31) plays diverse roles in vascular biology including monocyte transmigra- and IL-10 were determined by cytometric bead array. Platelet surface expression of tion, angiogenesis, platelet function, and thrombosis. Thus, PECAM-1 represents an PAC-1, P-Selectin and GPIb was significantly reduced after PCI in patients receiv- important link between leukocyte migration and angiogenesis. In the present study ing bivalirudin as compared to heparin. Similarly CD11b expression on CD14+ we investigated the effect of an endothelial cell-specific deletion of VEGF-R2/flk- monocytes was diminished after bivalirudin. Yet, no differences in cytokine levels 1, an important regulator of angiogenesis, and deletion of PECAM-1 in a murine between the bivalirudin and the heparin group before or after PCI were observed.
model of stagnant flow venous thrombosis.
Conclusion: Our data suggest that bivalirudin may reduce platelet and monocyte
Methods: Thrombosis was induced in the infrarenal vena cava of Tie2/Cre Flk-1
activation in patients undergoing elective PCI. Thereby, bivalirudin might reduce flox/flox mice on a C57/BL6 background and PECAM-1 -/- on a FVB/n background periinterventional thrombotic complications.
by creating a venous stenosis with a silk suture. Thrombi were harvested on days 3,7, 10, 14 and 28 after surgery (n=8 per time point). Non-transgenic siblings served as controls. Trichrome and immunohistochemical staining were performed.
Effect of Drotregocin alpha (activated) of sRAGE levels during the time course of
Results: Thrombus cross-sectional area analysis over time demonstrated a sig-
patients with severe sepsis
nificant increase in thrombus area by day 7 after surgery in flk-1-/- and by day 3 Hoffmann U1, Bopp C2, Hofer S2, Lang S1, Liebetrau C1, Saur J1, Borggrefe M1, Weigand M2 in PECAM-1 -/- animals compared with controls. Immunohistochemical staining using an antibody against antigen F4/80 for detecting macrophages in the thrombi st Medical Department, Faculty Of Clinical Medicine Mannheim, University Heidelberg, Germany revealed a decreased number of monocytes in flk-1-/- animals on day 7 and at all 2Department of Anesthesiology University of Heidelberg, Germany Background: The receptor for advanced glycation end products (RAGE) is a mem-
Conclusion: Cell-specific deletion of VEGF-R2/flk-1 and deletion of PECAM-1
ber of the Ig superfamily and a multiligand receptor interacting with a diverse class lead decreased monocyte numbers in thrombi and result in misguided thrombus of ligands. RAGE has secretory isoforms referred to as soluble RAGE (sRAGE), resolution. The data demonstrate that both endothelial Flk-1 and PECAM-1 might which comprise the extracellular ligand-binding domain but are lacking the cyto- play a role in monocyte recruitment into resolving thrombi.
solic and transmembrane domains. In humans, endogenous sRAGE is producedby alternative splicing of RAGE mRNA. We measured levels of sRAGE in septicpatients treated with and without Drotregocin alpha (activated) in septic patients.
Congress Program: 53rd Annual Meeting - Society of Thrombosis and Haemostasis Research Tagungsprogramm: 53. Jahrestagung der Gesellschaft für Thrombose- und Hämostaseforschung e.V. - GTH Patients and Methods: Blood samples were obtained from septic patients (n=8;
control-group) and septic patients under treatment with Drotrecogin alpha (acti-
vated) (n=8) on days 1, 3 and 5 of severe sepsis. SRAGE levels were measured by
ELISA-methods. Unpaired and paired Student`s t-test and Spearman correlation
were used to compare results.
Results: SRAGE levels decrease from day 1 (baseline) (Mean= 1,61 ng/ml +/-
SEM=0,50) to day 5 (Mean = 0,29 ng/ml +/- SEM=0,09 ) in septic patients treated
with Drotregocin alpha (activated) and were significantly lower on day 5 as com-
pared to day 1 (p<0,05). In septic patients not treated with Drotrecogin alpha (acti-
vated) (control-group) there was no significantly decrease of sRAGE levels from
day 1 (Mean=0,38 ng/ml +/- SEM=0,01) up to day 5 of sepsis (Mean=0,26 ng/ml
+/- SEM=0,05).
Conclusion: We can show that Drotrecogin alpha (activated) significantly decreases
sRAGE serum levels during the time course of septic patients. This observation
strongly suggests that Drotrecogin alpha (activated) plays an important role in
regulating the cellular damage during sepsis.
Inflammation and endothelial dysfunction in children with type 1 diabetes mellitus
Hörtenhuber T1, Rami R2, Elhenicky M1, Höbaus C1, Schober E2, Schernthaner G3, Schernthaner G
1Medical University Of Vienna, Internal Medicine II, Vienna, Austria, 2Medical University Of Vienna,
Pediatrics And Adolescent Medicine, Vienna, Austria, 3Rudolfstiftung Vienna, Medicine I, Vienna,
Objectives: Despite dramatic improvements in diabetes management, the macrovas-
cular disease (MVD) morbidity and mortality in type 1 diabetes mellitus (T1DM)
stayed elevated resulting in a loss of life of up to 20 years. Since the mechanisms are
not completely understood, we investigated inflammation and endothelial dysfunc-
tion in children with T1DM in comparison to healthy controls (CO).
Design and Methods: We included 175 children, 140 T1DM, 35 CO. As a general
marker of systemic vascular inflammation, we used C-reactive protein (CRP), as
early marker of endothelial dysfunction, endothelial progenitor cells (EPC). EPC
circulate in the peripheral blood, are involved in neovascularisation, endothelial
dysfunction and predict MVD morbidity and mortality. EPC (CD34+/CD133+/
CD309+) were enumerated by direct immunostaining technique and recorded on
a flow cytometer.
Results: Patients (T1DM) and controls (CO) were matched for age and
gender. EPC were significantly impaired in children with T1DM vs. CO
(609±359vs.1137±504,p<0.001). Other differences were found in systolic blood
pressure (115±15vs.101±8 mmHg,p<0.001), LDL-cholesterol (90±28vs.83±15mg/
dl,p=0.03), glucose (154±83vs.90±8mg/dl,p<0.001), CRP (112±14vs.101±8mg/
dl,p<0.001), each T1DMvs.CO. Multivariate regression revealed that HbA1c was
the strongest predictor of EPC in our cohort (beta=-0.355,p<0.001). Remarkably,
EPC and CPR were not associated with one another.
Conclusions: In conclusion, this is the first study investigating both inflammation
and endothelial dysfunction (by EPC measurements) in children with T1DM: EPC
are significantly reduced and CRP levels are significantly elevated. The missing
association of CRP and EPC in our study cohort, suggests a from inflammation
independent demetrial effect on EPC in children with T1DM.
Thrombotic Disorders and Antithrombotic Therapy
Congress Program: 53rd Annual Meeting - Society of Thrombosis and Haemostasis Research Tagungsprogramm: 53. Jahrestagung der Gesellschaft für Thrombose- und Hämostaseforschung e.V. - GTH


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