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Combined pancreas-kidney transplantation is one therapeutic option of Diabetes mellitus. In the early postoperative period pancreatic venous thrombosis is one of the major complications leading to allograft dysfunction and graft loss. One risk factor is the ischemia-reperfusion(I/R)- injury. Additionally, immunosuppressive drugs, such as the calcineurine inhibitor tacrolimus, have been implicated in the development of microvascular thrombosis. Several studies report a beneficial effect of antithrombin III (ATIII)- and N-acetylcysteine-(NAC)-substitution on the ischemia-reperfusion injury. Aim of this study was to analyse the therapeutic effect of ATIII and NAC on microvascular thrombus formation in post-ischemic tissue under immunosuppressive therapy with tacrolimus in vivo.

Models of skin graft revascularization are mostly based on histological evaluations, but lack the possibility to analyze the vascular biology in vivo. The aim of the present study was therefore to develop an animal model which allows continuous monitoring of the microcirculation during skin graft healing. Skin and subcutaneous tissue were removed from the back of dorsal skinfold chamber preparations in mice, leaving one layer of striated muscle and subcutaneous tissue as wound bed. A respective full-thickness skin graft was harvested from the groin and sutured into the defect in the back of the chamber. To study graft healing, repetitive intravital microscopy was performed during the first 10 days after engraftment. Capillary widening in the wound bed appeared at day 1 after grafting and increased until day 4. Capillary buds and sprouts firstly appeared at day 2. Blood filling of autochthonous graft capillaries occurred at day 3, resulting in almost complete restoration of the original skin microcirculation at day 5. This was achieved by interconnections between the microvasculature of the wound bed and the skin graft through a temporary angiogenic response. In principle, angiogenic blood vessel growth originated in the wound bed and was directed towards the graft. This new model allows for repetitive analysis of the microcirculation during skin graft healing. It provides ideal in vivo conditions to further delineate the exact mechanisms of blood vessel interconnection during the complex process of angiogenesis, and may also allow to study the vascularization of tissueengineered skin substitutes.

Thrombotic microangiopathy (TMA) is a fatal complication that occurs after combined pancreaskidney transplantation leading to allograft dysfunction and graft loss. Immunosuppressive drugs, such as calcineurin inhibitors, have been implicated in the development of nonimmune TMA. Tacrolimus has been shown to be associated with decreased NO-production, thus contributing to thrombus formation. ADMA acts as an endogenous NO-synthase inhibitor and is considered to be a marker of endothelial dysfunction. Aim of this study was to analyse the influence of different immunosuppressants on microvascular thrombus formation in vivo and to further examine the underlying endothelial function. Using the skin fold chamber in C57BL/6J mice, microvascular thrombus formation was induced photochemically and was quantitatively analyzed by intravital fluorescence microscopy. Mice were treated with tacrolimus (TAC: 10 mg/kg/d; n = 7), cyclosporine (CYA: 5 mg/kg/d) or sirolimus (RAPA: 1,5 mg/kg/d ip) on 3 consecutive days. Control mice received NaCl 0,9 % (10 ml/kg/d ip; n = 5). Drug plasma levels were examined to ensure therapeutic doses. Additionally, the systemic circulation of sP- and sE-selectin as well as ADMA plasma levels were measured by ELISA. Application of immunosuppressants produced clinically relevant plasma levels (TAC: 8 ± 2 ng/ml, CYA: 214 ± 16 ng/ml; RAPA: 9 ± 1 ng/ml). Microvascular thrombus formation was significantly accelerated in mice receiving TAC compared to control mice (arteriolar and venular occlusion: 251 ± 101s and 179 ± 27 s vs. control 818 ± 221 s and 749 ± 231 s; p < 0,05). Application of CYA significantly increased thrombus formation only in venules (276 ± 49s; p < 0,05 vs. control), whereas RAPA had no significant effect on thrombosis induction. Plasma concentrations of sP-Selectin and sE-selectin were slightly reduced after TAC and CYA application. ADMA-levels however were significantly increased in mice receiving TAC (1,28 ± 0,16 µmol/l vs. control: 0,74 ± 0,13 µmol/l; p < 0,05). The present results show that clinically relevant plasma levels of cyclosporine enhanced thrombus formation only in venules whereas tacrolimus significantly accelerates thrombus formation in arterioles and venules. This effect could be explained by increased ADMA-levels in plasma resulting in impaired NO-bioavailability and increased thrombogenicity, thus presenting a new molecular mechanism in the development of transplant-thrombosis.

Erythropoietin-treatment is generally assumed to be associated with an increased risk for thrombosis due to an elevated hematocrit. However, previous in vitro and in vivo studies of our group revealed a reduction of platelet reactivity and no significant effect on microvascular thrombus formation during treatment with darbepoetin-alpha (DPO), an erythropoietin-derivative with a 3-fold longer half life. Aim of the study was therefore to investigate the effect of DPO application on endothelial function and to further characterize the role of eNOS in this context.

Cigarette smoking increases the risk for myocardial diseases and is associated with a higher incidence of postoperative complications. Previous studies showed an increased leukocyte rolling and adhesion following nicotine application. Next to this the use of estrogen containing oral contraceptives is linked to a higher risk for deep vein thrombosis. However, clinical studies investigating the influence of transdermal nicotine application on cardiovascular risk factors could not show a negative effect of nicotine. Therefore transdermal nicotine replacement is regarded as a safe aid to smoking cessation. It is still not clear whether nicotine or other substances in cigarette smoke are responsible for the prothrombotic risk associated with cigarette smoking. Aim of this study was therefore to investigate the influence of nicotine on microvascular thrombus formation in vivo and to further characterize the underlying mechanisms. In microvessels of dorsal skin fold chambers of mice, light-dye-induced thrombus formation was analyzed using intravital fluorescence microscopy. Male and female C57BL/ 6J mice received nicotine chronically via the drinking water (100 µg/ml) for 8 weeks. Control animals of both gender received normal drinking water. Subsequently plasma levels of cotinine, a stable metabolite of nicotine, were determined. To study the influence of nicotine on endothelial cell function immunohistochemical tissue sections for the vascular localization of P-selectin, PAI-1, PAF-R and ICAM-1 were performed. Furthermore the plasma concentrations of soluble P-selectin, E-selectin, ICAM-1 and VCAM-1 were quantified by ELISA. To characterize the influence of nicotine on platelet activation, flowcytometric analyses of the expression of P-selectin, GPIIb/IIIa and CD107a on murine platelets were carried out. Chronic nicotine exposition caused cotinine plasma levels similar to those seen in chronic smokers (> 100 ng/ml vs. controls: < 10 ng/ml). In male animals no significant differences in microvascular thrombus formation were detected following chronic nicotine application, when compared to controls (complete arteriolar and venular occlusion: 640 ± 96 s and 427 ± 51 s vs. control: 577 ± 78 s and 531 ± 102 s). Likewise in female animals no significant differences were found, following long-term nicotine application (complete arteriolar and venular occlusion: 715 ± 130 s and 493 ± 79 s vs. control: 866 ± 24 s and 481 ± 70 s). In line with this, flow cytometric analyses did not show significant effects of nicotine on P-selectin, GPIIb/IIIa and CD107a expression on resting platelets or upon stimulation with thrombin (20 U/ml). In contrast to this chronic nicotine application caused a marked reduction of vascular expression of P-selectin, PAF-R and PAI-1 in male mice, while in female mice only PAI-1 was found significantly reduced. Additionally chronic nicotine treatment lead to a significant reduction of sP-selectin and sE-selectin circulation in male mice. The present study shows no significant influence of chronic nicotine treatment on microvascular thrombus formation and platelet adhesion molecule expression despite elevated cotinine levels in mice of both gender. However, chronic nicotine treatment resulted in a significant reduction of endothelial adhesion molecule expression in male mice, while there was only a slight effect in female mice.

In der vorliegenden Arbeit konnte unter Nutzung zweier unterschiedlicher Versuchsmodelle eindrucksvoll gezeigt werden, dass bei septischen Bedingungen der hochdosierte Einsatz einer alleinigen AT-Gabe (250 U/kg) der klassischerweise mit Heparin durchgefuhrten Thromboseprophylaxe uberlegen ist. Allerdings kam es unter diesem Regime zu einer Verlangerung der Blutungszeit. Erstmalig konnte somit in zwei gut etablierten experimentellen Sepsismodellen die antithrombotische Potenz von Antithrombin zur Verhinderung der mikrovaskularen Thrombosierung charakterisiert werden. Damit konnte in der adjuvanten Therapie septischer Patienten auf die Heparingabe verzichtet werden zugunsten von AT und dessen anti-inflammatorischen Potenz.

Die vorliegende Studie zeigt bei masig erhohten, Darbepoetin-α induzierten Hamatokritwerten von ca. 55 % bereits eine reduzierte Aktivierbarkeit von Thrombozyten im Sinne einer Zelldysfunktion, welche sich jedoch nicht in einer Blutungsneigung mit Verlangerung der mikrovaskularen Thrombusbildung ausert. Zur endgultigen Klarung des Effektes von Erythropoietin bzw. seiner Derivate auf die Hamostase bedarf es weiterfuhrender Analysen endothelialer und plasmatischer Faktoren.

Ebselen, a seleno-organic compound showing glutathione peroxidase-like activity, has potent anti-inflammatory and anti-oxidant effects. Since selenium deficiency is thought to be associated with an increased incidence of vascular thrombosis, we studied the effect of ebselen on blood cell aggregate formation and vessel occlusion in vivo. In individual microvessels of rat cremaster muscle preparations photochemically induced thrombus formation was analyzed in detail using intravital fluorescence microscopy. In ebselen-pretreated animals (30 mg/kg ip), venular thrombus formation was significantly delayed (50% vessel occlusion: 535 ± 34s; initial stasis: 872 ± 82s; complete occlusion: 908 ± 87s) as compared to vehicle-treated controls (416 ± 42s; 612 ± 49s; 647 ± 51s). Moreover, ebselen significantly prolonged the kinetics of arteriolar thrombus formation and even completely prevented blood cell aggregate and thrombus formation in 88.9% of all arterioles studied (p < 0.05 vs controls: 37.5%). As assessed by flow cytometry, oxidant stress-induced upregulation of CD62P on isolated platelets was found dose-dependently inhibited by increasing concentrations of ebselen (10 – 100 μM), suggesting that the antithrombotic effect of ebselen is achieved by attenuation of CD62P-dependent platelet/leukocyte aggregation. Thus, ebselen represents preventive and therapeutic value for disorders with increased risk for oxidant stress-associated thrombotic events.