Thilo Schäfer

Local Heat Shock Priming Promotes Recanalization of Thromboembolized Microvasculature by Upregulation of Plasminogen Activators

Objective—Thromboembolization and subsequent microvascular perfusion failure is implicated in the pathology of a variety of diseases, including transient ischemic attack (TIA), stroke, and myocardial infarction, and also for the complications after interventional and microsurgical procedures in coronary heart disease and peripheral arterial occlusive disease. In vitro heat shock priming has been suggested to induce plasminogen activators, which are the major upregulators of the fibrinolytic system. Herein, we determined whether local heat shock priming endogenously upregulates plasminogen activators also in vivo, and whether this promotes recanalization of thromboembolized microvasculature. Methods and Results—To induce thromboembolization, a suspension of preformed microthrombi (maximum diameter: 40 &mgr;m) was injected via the femoral artery into the left hindlimbs of anesthetized rats. Local heat shock priming (42.5°C, 30 minutes) was performed 24 hours before embolization and resulted in a significant increase of endothelium-derived plasminogen activator expression. The study of the microcirculation by intravital microscopy revealed in all tissues analyzed (muscle, periosteum, subcutis, and skin) that heat shock priming significantly (P<0.05) accelerates recanalization of the thromboembolized microvasculature when compared with nonprimed and sham-primed controls. Importantly, the addition of plasminogen activator inhibitor-1 to the microthrombi suspension completely blunted the heat shock-induced acceleration of microvascular recanalization. Conclusions—Heat shock induces endogenous hyperfibrinolysis by upregulation of plasminogen activators that promote recanalization of thromboembolized microvasculature.

Die hepato-arterielle Infusion von rh-TNF-α induziert ein kapillares Perfusionsversagen und apoptotischen Zelltod in Lebermetastasen kolorektaler Karzinome

Background: The application of TNF-α by hepatic-arterial infusion (HAI) might be an option in the treatment of patients with liver metastases from colorectal carcinoma. The mechanisms of action, however, are still not completely clarified. Herein, we studied specific TNF-effects on hepatic tumor microcirculation. Methods: 21 WAG/RIJ rats were randomized in three groups. 11 days after hepatic implantation of 5 × 105 CC531 colorectal cancer cells, animals underwent HAI with 15 µg/kg (n = 7) or 30 µg/kg (n = 7) of rh-TNF-α. Saline group was used for control (n = 7). The microcirculation of liver and tumor tissue was analyzed in vivo by quantitative multi-fluorescence microscopy. Also, blood samples were asserved. Results: Initially after TNF-HAI we found a massive perfusion failure in tumor capillaries, whereas there was an only slight alteration of tumor microcirculation in the control group. Of interest, rh-TNF-α did not affect sinusoidal perfusion of the tumor neighbouring liver tissue. The TNF-α-induced occlusion of tumor capillaries was associated with an additional decrease of red blood cell velocity in tumor venules. Further, we detected an overall pro-inflammatory cytokine response. This was accompanied by an aggravation of leukocyte-endothelial cell interaction in tumor venules and a caspase-3-dependent apoptotic cell death. Discussion: Thus, we found that TNF-α-based HAI induces selective deterioration of tumor microcirculation with accumulation of leucocytes. The microvascular perfusion failure has to be considered as the cause for apoptotic cell death, which seems to be one of the mechanism of selective tumor destruction after regional therapy with TNF-α.

Reduktion der Endotoxinämie-induzierten inflammatorischen Antwort und hepatozellulären Apoptose durch in vivo Blockade von p53

Sepsis-associated multiple organ failure still represents a challenging disease with high mortality. Thus far, experimental studies have shown that endotoxinemia causes parenchymal organ dysfunction via the release of cytokines, the activation of leukocytes and alteration of the microcirculation with, finally, apoptotic cell death. Therefore the aim of our study was to evaluate whether a temporary blockade of p53 with pifithrin-α is capable to reduce apoptotic cell death and organ dysfunction in LPS-induced endotoxinemia in rats. Male Sprague-Dawley rats were either treated with pifithrin-α (PFT-α, 2.2 mg/kg bw, ip, n = 7) or with the vehicle (DMSO, 1 mg/kg bw, control, n = 11) 30 min before induction of endotoxinemia (LPS 10 mg/kg bw, iv). After 16 h of endotoxinemia, animals underwent anesthesia and median laparotomy. The common bile duct was cannulated and the left liver lobe was exteriorized for intravital microscopy. After i.a. injection of fluorescent dyes, hepatocellular apoptosis (bisbenzimide, H33342,2 μmol/kg), sinusoidal perfusion (Na-fluoresceine, 2 μmol/kg), leukocyte adherence (rhodamin 6G, 0.1 μmol/kg) and Kupffer cell phagocytic activity (fluorescently labelled latex particles, 3×108/kg) were analysed. Bile flow served as an indicator for hepatocellular excretory function. After 16 h of endotoxinemia, PFT-αpretreatment caused a significant decrease of the mortality rate and a reduction of the number of apoptotic hepatocytes. Moreover PFT-α treatment caused a significant decrease of the leukocytic inflammatory response with, concomitantly, a significantly higher percentage of perfused sinusoids and increased bile production. Finally, PFT-αnormalised the endotoxinemia-associated depression of Kupffer cell phagocytic activity. The present study demonstrates for the first time attenuation of mortality rate, apoptotic cell death and organ dysfunction by PFT-αin a rat model of endotoxinemia. Thus, temporary p53-inhibition seems to be a promising novel tool in the treatment of septic shock conditions.

Reduktion der inflammatorischen Antwort nach Lappentransfer durch lokale Hitzeschock-Vorbehandlung

Der Transfer freier mikrochirurgischer Lappen beinhaltet Ischamie und anschliesende Reperfusion. Dadurch konnen inflammationsbedingte Mikrozirkulationsschaden entstehen [1]. Bisherige Studien zeigten, das Streskonditionierung durch Induktion von Hitzeschockproteinen (HSP) zu einer temporaren Gewebeprotektion fuhrt [2]. Inwieweit dabei die inflammatorische Antwort beeinflust wird, ist bisher unbekannt. Wir untersuchten daher anhand eines freien osteomyokutanen Lappens, inwieweit lokale Hitzeschockvorbehandlung uber die Wirkung von HSP-32 (identisch mit Hamoxygenase-1) [3], zu einer Reduktion der inflammatorischen Antwort nach Lappentransfer fuhrt.