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

【摘要】  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 µm) 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.

Using a rat thromboembolization model and intravital fluorescence microscopy, this study demonstrates that heat shock priming induces endothelium-derived plasminogen activator expression in vivo, and accelerates spontaneous recanalization of thromboembolized microvasculature. This was completely blunted by application of plasminogen activator inhibitor-1. Thus, heat shock induces endogenous hyperfibrinolysis, which promotes recanalization of thromboembolized microvasculature.

【关键词】  heat shock intravital microscopy microcirculation plasminogen activator inhibitor thromboembolization urokinase plasminogen activator

Introduction

In myocardial ischemia and stroke, thromboembolization of the microvasculature essentially contributes to infarction and necrosis. 1 In addition, vascular interventional procedures, such as PTA 2 and PTCA, 3 but also coronary bypass surgery 4 and peripheral reconstructive microsurgery, 5 may be complicated by downstream microvascular thromboembolization with the consequence of nutritive capillary perfusion failure.

Recanalization of thromboembolized microvasculature can be achieved by urokinase therapy; however, this bears some risk for bleeding and aggravation of organ dysfunction, particularly if a surgical procedure is involved. 6 In contrast, endogenous induction of hyperfibrinolysis would represent a more elegant approach; however, little information is available as to whether this may be capable of successfully promoting recanalization of thromboembolized microvasculature.

Preconditioning by heat shock requires the exposure of the tissue to a supraphysiological but sublethal temperature, which results in a transient change of cellular biosynthesis with an accelerated induction of only a few distinct proteins, including heat shock proteins. 7

The serine proteases urokinase plasminogen activator(uPA) and tissue-type plasminogen activator (tPA) initiate the endogenously mediated lysis of platelet arterial emboli. Both enzymes derive from endothelial cells and convert plasminogen to the fibrinolytic protease plasmin. 8 Various types of stimuli are known to directly modulate plasminogen activator synthesis and release from endothelial cells. 9,10 In vitro, stress conditioning by heat shock has been shown to induce plasminogen activators in human umbilical vein endothelial cells. 11 With the use of a rat hindlimb microcirculation model, we herein demonstrate for the first time to our knowledge that local heat shock priming induces upregulation of plasminogen activators also in vivo, and that this promotes endogenous hyperfibrinolysis, which results in accelerated recanalization of thromboembolized microvasculature.

Methods

Animals

Experiments were performed in 72 Sprague-Dawley rats with a body weight of 280 to 350 grams. The study complied with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and was approved by the local animal care committee.

Surgical Procedure

Under pentobarbital anesthesia (50 mg/kg intraperitoneal; Abbott, Chicago, Ill), the animals underwent tracheotomies and were placed on a heating pad to guarantee 37°C body temperature. Polyethylene catheters were inserted into the right carotid artery and left jugular vein. The catheters allowed monitoring of blood pressure, continuous infusion of saline (1 mL/100 g per hour), withdrawal of blood, and injection of fluorescent dyes for intravital microscopy.

The left hindlimb preparation for microcirculatory analysis was performed according to the technique described previously in detail. 12 The preparation exposed tibial periosteum, gracilis and semitendinosus muscles, subcutis and skin, and was supplied by the femoral vessels. All branching vessels were ligated up to the superficial epigastric artery, in which a catheter was inserted with the tip directed toward the femoral artery.

Microvascular Thromboembolization

Arterial platelet-rich thrombi were preformed in vitro in a moving high-pressure closed compartment system. 13 For each experiment a 1-mL insulin syringe was filled with thrombin (60 U/mL; T6634; Sigma-Aldrich, Taufkirchen, Germany). In a second syringe, 400 µL blood was drawn from the carotid artery. After 20 seconds, the 2 syringes were interconnected and the suspension containing blood and thrombin (4:1) was moved during 3 minutes &70 times from one syringe to the other. In additional experiments, a rat-specific plasminogen activator inhibitor (PAI)-1 (30 pg/mL; #102; American Diagnostica, Pfungstadt, Germany), the principal physiological inhibitor of both tPA and uPA 14 was added to the microthrombi suspension. The syringes were left standing for 30 minutes until embolization. 40 µm, the thrombi suspension was filtered through a nylon strainer (2340; Falcon; Becton Dickinson, Heidelberg, Germany). For thromboembolization of the microvasculature in vivo, 30 µL of the filtered thrombi suspension was gently injected into the femoral artery. 15

Intravital Fluorescence Microscopy

The tissues were positioned on a micromanipulator-adjusted stage and covered with a glass slide to prevent drying and exposure to ambient air. The microcirculation was analyzed with a blue 520 nm) after intravenous injection of 5% fluorescein isothiocyanate (FITC)-labeled dextran (MW 150 000; Sigma-Aldrich) using a modified epi-illumination Axiotech microscope (Zeiss, Jena, Germany). The contrast enhancement achieved guarantees high-resolution imaging of the microcirculation. 16

Video Analysis

Microscopic images were recorded by a charge-coupled device video camera (FK-6990, COHU, Prospective Measurements, San Diego, Calif) and transferred to a video system. Microcirculatory parameters were quantified off-line using a computer-assisted image analysis system (Capimage, Zeintl, Heidelberg, Germany). After thromboembolization, microvascular recanalization was analyzed by studying the re-onset of capillary perfusion. In addition, a 4-hour follow-up assessment included the determination of: (1) the fraction of perfused capillaries (%), defined as percentage of the length of red blood cell (RBC) perfused capillaries relative to the total length of all capillaries per unit area; (2) capillary V RBC (mm/s); and (3) capillary diameters (µm). 16 Volumetric capillary blood flow (CBF) (pL/s) was calculated for each microvessel as CBF= *(D/2) 2 *V RBC.

Stress Conditioning

For stress conditioning, left hindlimbs of anesthetized rats were heated in a waterbath 24 hours before thromboembolization. 17 During local heating, muscle temperature was increased to 42.5°C and was kept constant for 30 minutes. Animals undergoing a sham heating procedure were also anesthetized 24 hours before thromboembolization, and the hindlimb was exposed in a waterbath to 30°C for 30 minutes. In these animals, the muscle temperature was kept at &36°C to 37°C. Muscle temperature was monitored with a needle thermo-probe (LICOX-System; GMS, Kiel-Mielkendorf, Germany).

Immunohistochemistry

After elimination of endogenous peroxidase activity and nonspecific protein binding, specimens were incubated overnight at 4°C with either anti-uPA-antibody (1:200; #1191; American Diagnostica), anti-tPA-antibody (1:200; kindly provided by J.J. Emeis; Leiden, Netherlands) or anti-PAI-1-antibody (1:200; #1062; American Diagnostica), which react specifically with rat uPA, rat tPA and rat PAI-1, respectively. As secondary antibody, either a biotinylated donkey-anti-chicken-IgG (Dianova, Hamburg, Germany) or goat-anti-rabbit-IgG antibody (DAKO-Cytomation, Hamburg, Germany) was used. Thereafter, streptavidin-horseradish peroxidase complex was added for 30 minutes at 20°C, followed by 5 minutes of treatment with 3,3'diaminobenzidine.

Slides were counterstained with hematoxylin. For negative controls, slides were treated similarly but without the primary antibody. All control stainings were negative.

The staining intensity was evaluated blindly according to Page et al 18 A "0 to 4" score was applied, depending on color intensity and extent, ie, 0 indicates negative, comparable to control section; 1, very weakly positive; 2, weakly positive; 3, positive; and 4, strongly positive.

Western Blot Analysis of uPA, tPA, and PAI-1

Muscle and skin tissue (n=4 per group) of control and heat shocked animals was harvested after 24 hours and homogenized in lysis buffer (10 mmol/L Tris pH 7.5, 10 mmol/L NaCl, 0.1 mmol/L EDTA, 0.5% Triton-X-100, 0.02% NaN 3, 0.2 mmol/L phenyl-methyl-sulfonyl-fluoride), incubated for 30 minutes on ice, and centrifugated for another 30 minutes at 16 000 g and 4°C. Equal amounts of protein per lane (90 µg) were separated discontinuously on 10% sodium dodecyl sulfate polyacrylamide gels under denaturing conditions and transferred to a polyvinyldifluoride membrane (BioRad, Munich, Germany). After blockade of nonspecific binding sites, membranes were incubated for 2 hours with a chicken-anti-rodent-uPA (1:50; American Diagnostica), a goat-anti-rat-tPA (1:50; Santa Cruz Biotechnology) or a rabbit-anti-rat-PAI-1 antibody (1:50; American Diagnostica) followed by the secondary horseradish peroxidase-conjugated bovine-anti-chicken (1:5000; Santa Cruz Biotechnology), rabbit-anti-goat (1:2000; R&D, Wiesbaden, Germany), and donkey-anti-rabbit (1:2500; Amersham Biosciences, Freiburg, Germany) IgG antibodies, respectively. Protein expression was visualized using luminol-enhanced chemiluminescence and exposure of membranes to blue light-sensitive autoradiography film. Signals were densitometrically assessed and normalized to ß-actin signals to correct unequal loading.

Experimental Groups

In a first group of animals (n=8), stress conditioning by local heat shock priming was performed 24 hours before thromboembolization. Animals undergoing a sham procedure of local heat shock priming (n=4) and animals without stress conditioning (n=8) served as controls. In an additional control group, the normal microcirculation was studied without thromboembolization and without treatment (n=8), and a further group of animals was designed to study the effect of heat shock preconditioning alone without thromboembolization (n=8). To study the role of plasminogen activators, further heat shock-primed (n=8) and nonheat shock-primed animals (n=8) underwent thromboembolization with PAI-1-supplemented microthrombi suspensions. The microcirculation of muscle, periosteum, subcutis and skin was analyzed before and at 30 minutes, 60 minutes, 120 minutes, 180 minutes, and 240 minutes after thromboembolization. Tissue samples for immunohistochemistry were obtained at thromboembolization (additional animals) and 4 hours recanalization.

Statistical Analysis

Results are expressed as means±SEM. Differences between groups were assessed by 1-way ANOVA, differences within each group were analyzed by 1-way repeated measures ANOVA. To isolate overall differences, appropriate Student-Newman-Keuls or Dunn post-hoc tests were performed. Differences were considered significant at P <0.05.

Results

Systemic Circulatory Parameters 100 mm Hg) and heart rate (340 to 400 minutes -1 ) were in normal range without significant differences between the groups studied.

Expression of uPA, tPA, and PAI-1

In nonheat shock-primed controls, immunohistochemistry revealed almost lack of expression of uPA and an only slight expression of tPA, which was found predominantly localized in arteriolar endothelial cells ( Figure 1 ). After heat shock, expression of uPA and tPA was significantly enhanced ( Table ), predominantly in endothelial and smooth muscle cells ( Figure 1 ). PAI-1 increased slightly on heat shock-priming but was less pronounced compared with uPA and tPA ( Table; Figure 1 ). In all experiments, uPA, tPA, and PAI-1 expression was not affected by adding PAI-1 to the microthrombi suspension.

Figure 1. Expression of uPA (A,B,C), tPA (D,E,F), and PAI-1 (G,H,I) in subcutaneous arterioles 4 hours after thromboembolization without heat shock priming (A,D,G), after heat shock priming (B,E,H), and after heat shock priming and PAI-1 application (C,F,I). Note that after heat shock priming, uPA (B,C) is predominantly expressed in endothelial cells (arrowheads), smooth muscle cells (arrows), and fibroblasts (asterisk), and tPA (E,F) mainly in endothelial cells (arrowheads). Nonheat shock-primed controls reveal almost lack of uPA expression (A), and only weak expression of tPA in endothelial cells (D). PAI-1 was only slightly increased after heat shock in endothelial cells (arrowheads) compared with nonheat shock-primed controls. Magnifications x 160 (A,B,C,G,H,I) and x 230 (D,E,F).

Immunohistochemistry of uPA, tPA, and PAI-1 in Arterioles of Muscle and Subcutis at Thromboembolization and 4-Hour Recanalization

Quantitative Western blot analysis confirmed a significant upregulation of uPA and tPA at 24 hours after heat shock preconditioning, whereas PAI-1 expression was only slightly increased in muscle and not affected in skin compared with nonheat shocked controls ( Figure 2 ).

Figure 2. Western blot analysis of PAI-1, uPA, and tPA in muscle (A,B) and skin (C) at 24 hours after heat shock preconditioning (hs) (n=4) and in nonheat shock-primed controls (con) (n=4). Representative examples from muscle (A) and quantitative analysis from muscle (B) and skin (C). Note the significant increase of uPA and tPA after heat shock priming, whereas PAI-1 was only slightly increased. Mean±SEM; * P <0.05 vs nonheat shock-primed controls.

Microvascular Thromboembolization

Directly after injection, intravital microscopy revealed that the microthrombi were arrested in the downstream microcirculation ( Figure 3 ). This resulted in a complete shutdown of microvascular perfusion in all tissues analyzed.

Figure 3. Intravital microscopy of thrombolysis in a transverse arteriole. The thrombus (arrows) is visualized by negative contrast using FITC-dextran plasma staining (A) and directly by rhodamine-staining of platelets (B). C and D, Identical arteriole after onset of recanalization. Magnification x 80. E and F, Rate and time course of recanalization of microthrombi (E, n=8) and microthrombi containing PAI-1 (F, n=8). Note the acceleration of recanalization by heat shock preconditioning (closed circles) compared with nonpreconditioned (open circles) and sham-preconditioned (open diamonds) controls.

Microvascular Recanalization

During the first 30 minutes after thromboembolization, nonheat shock-primed and sham heat shock-primed controls showed almost complete lack of recanalization. Only 1 of 8 and 1 of 4 preparations revealed signs of recanalization ( Figure 3 ). During the second 30 minutes after thromboembolization, all preparations developed recanalization ( Figure 3 ), but with different quality of microvascular reperfusion. After 30 minutes only 10% of the initially perfused capillaries were found reperfused ( Figure 4 ). Recanalization improved during the next 2 hours, as indicated by an increase of perfused capillaries to &50% of baseline, but without further recovery during the 4-hour observation period ( Figure 4 ). This was associated with a markedly lowered volumetric CBF ( P <0.05; Figure 5 ). There were no differences in recanalization between muscle, periosteum, subcutis and skin, and no significant differences between nonheat shock-primed and sham heat shock-primed animals ( Figure 4 ).

Figure 4. Fraction of perfused capillaries in nonheat shock-primed (open circles), sham heat shock-primed (open diamonds) and heat shock-primed (closed circles) muscle (A,B,C), skin (D,E,F), subcutis (G,H,I), and periosteum (J,K,L) before and after thromboembolization with microthrombi (B,E,H,K) and microthrombi containing PAI-1 (C,F,I,L). Preparations without thomboembolization served as controls (A,D,G,J). Mean±SEM; * P <0.05 vs nonheat shock-primed controls, # P <0.05 vs baseline.

Figure 5. Volumetric CBF in nonheat shock-primed (open circles), sham heat shock-primed (open diamonds) and heat shock-primed (closed circles) muscle (A,B,C), skin (D,E,F), subcutis (G,H,I), and periosteum (J,K,L) before and after thromboembolization with microthrombi (B,E,H,K) and microthrombi containing PAI-1 (C,F,I,L). Preparations without thromboembolization served as controls (A,D,G,J). Mean±SEM; * P <0.05 vs nonheat shock-primed controls, # P <0.05 vs baseline.

Heat shock priming effectively accelerated initial recanalization of the obstructed microvasculature. Within the first 15 minutes after thromboembolization, all 8 tissue preparations showed recanalization ( Figure 3 ). At 30 minutes, already 60% of the initially perfused capillaries were found reperfused in either of the tissues analyzed ( P <0.05; Figure 4 ). Recanalization over the subsequent 3.5-hours further improved capillary perfusion to &80% of baseline, which was significantly higher than that of nonheat shock-primed and sham-heat shock-primed controls ( P <0.05; Figure 4 ).

Importantly, heat shock-primed tissues showed already at baseline a significantly higher volumetric CBF ( P <0.05) than nonconditioned tissues ( Figure 5 ). Further, heat shock produced reactive hyperemia during initial recanalization and preserved CBF over the entire post-thromboembolization period.

Addition of PAI-1 to the thrombi resulted in prolonged failure of recanalization ( P <0.05). Until 2 hours after thromboembolization, zero and, at 4 hours only 2, of 8 preparations showed recanalization ( Figure 3 ). This was associated with a significantly ( P <0.05) lowered fraction of perfused capillaries ( Figure 4 ) and a marked ( P <0.05) compromise of CBF ( Figure 5 ) compared with nontreated controls. Heat shock priming accelerated the onset of recanalization and improved the microcirculation ( Figures 4 and 5; P <0.05), although the addition of PAI-1 was associated with a reduction of capillary density and CBF when compared with heat shock-conditioned but non-PAI-1-treated controls ( Figures 4 and 5; P <0.05).

Microvascular Response to Heat Shock Priming

In animals without any treatment, analysis of capillary perfusion over the 4-hour observation period showed &100% of the capillaries perfused without significant changes of CBF ( Figures 4 and 5 ). Heat shock priming without thromboembolization did not affect capillary density ( Figure 4 ) and confirmed the increased CBF compared with nonheat-shocked controls ( Figure 5 ).

Discussion

The major novel findings of the present study are that heat shock priming: (1) upregulates plasminogen activators in vivo; (2) accelerates recanalization of thromboembolized microvasculature; and (3) counteracts the function of PAI-1. Thus, local upregulation of heat shock proteins induces endogenous hyperfibrinolysis.

Thromboembolization significantly contributes to the shutdown of microvascular perfusion, infarction, and necrosis. 1,3 The microemboli may clot primarily arteriolar and capillary segments of the microvasculature, and thus produce initially a negative angiogram. 19 Over time, obstruction of the vasculature may aggravate, producing relevant infarction, or may dissolve because of spontaneous recanalization. 20,21

The study of the mechanisms of thromboembolization requires an adequate experimental model, which should consider the development of both infarction and spontaneous recanalization. O?Shaughnessy et al 22 introduced a thromboembolization model, demonstrating that platelet emboli, originating from the site of arterial vessel repair, pass downstream, and block the microcirculation. 22 This confirms the particular risk of interventional and microsurgical procedures for thromboembolism. Herein, we have not chosen this model, because the development of thromboemboli is heterogeneous, and the cremaster muscle allows only a 2-hour study period, which is too short to analyze spontaneous recanalization. We have used the rat hindlimb model, because this allows repetitive intravital microscopy of the microcirculation of muscle, periosteum, subcutis, and skin for a period of 6 hours. 12 Further, we have injected in vitro produced microthrombi, mimicking platelet-rich arterial thrombi, which adequately standardizes the experiments. 15

The process of platelet-rich arterial thrombus formation can be partitioned into platelet adhesion, coagulation factor activation and thrombus propagation. 23 Accordingly, in the model used thrombi were generated by addition of thrombin (coagulation factor activation). Because a low pressure in vitro system can produce large "white" thrombi, however, with a "red" tail, 24 we have chosen a moving high-pressure closed compartment system, 13 which generates arterial "white" thrombi, rich in platelets and fibrin, intermingled with only a few erythrocytes and leukocytes. 13 The moving high-pressure closed compartment system mimics conditions of pressure and turbulences as known in arterial thrombus formation in vivo. The platelet-rich thrombi were filtered through a 40-µm 40 µm. Only small thrombi of <40 µm were used for the experiment to guarantee that embolization takes place within terminal arterioles. This microembolization mimics the clinical situation of ischemic stroke and flap tissue failure. 5,13,22

Because terminal arterioles are not regularly visible by intravital microscopy, 15 recanalization was not assessed by direct visualization of the lysis of arrested microthrombi, but indirectly by evaluation of the re-onset of capillary perfusion within the downstream microcirculation. This can reliably be performed, because downstream capillary perfusion failure, which additionally indicates tissue viability, has been shown to correlate with embolization-induced arteriolar vessel obstruction. 25

Previous studies have demonstrated that heat shock increases capillary perfusion in flap tissue. 17 Thus, the increased baseline CBF may have contributed to the accelerated recanalization. However, although baseline skin CBF did not differ significantly between heat shock preconditioning versus non-preconditioned controls, heat shock significantly accelerated recanalization, similarly as observed in the other tissues, in which baseline CBF was different between the 2 groups. Thus, the heat shock-mediated increase in basal CBF may not be considered a primary determinant for recanalization.

Beside acceleration of recanalization and improvement of capillary perfusion, heat shock increased CBF above baseline levels initially during recanalization. This reactive hyperemia, which classically is produced only after short ischemia periods, may be caused by the rapid lysis-associated reduction of ischemia time by heat shock priming.

Because anesthesia and immersion of the hindlimb may also represent a stress conditioning, we have exposed additional animals to a sham procedure, which was not associated with tissue heating. Of interest, these animals did not show acceleration of recanalization and improvement of capillary perfusion. This supports that the protective effect on the microcirculation is caused by heat induction rather than anesthesia or hindlimb manipulation-associated stress.

By converting plasminogen to plasmin, plasminogen activators are the main upregulators of the fibrinolytic system. 8 The serine proteases uPA 26 and tPA 27 are synthesized by endothelial cells. In vivo, tPA is the principal plasminogen activator, whereas uPA serves as an amplifier of the tPA-mediated fibrinolysis after its activation by tPA-mediated generation of plasmin. 28 A procoagulant condition induced by thrombin is the predominant physiological stimulus for expression and release of both plasminogen activators. 29

In vitro studies have demonstrated that prolonged hyperthermia 8 hours downregulates plasminogen activators and stimulates PAI-1 expression in human umbilical vein endothelial cells. 30,31 However, hyperthermic exposure times of <8 hour indicated that both uPA and tPA are upregulated by heat shock in endothelial cells. 11 The present study extends this observation, demonstrating for the first time to our knowledge that 30 minutes of heat shock upregulates uPA and tPA also in vivo, and that the expression is found primarily in arteriolar endothelial cells. Thus, an enhanced lysis of fibrin in platelet emboli by uPA and tPA may be the cause for the observed acceleration of recanalization after heat shock priming.

Fibrinolysis is inhibited by endothelium-derived PAI-1, which binds irreversibly to the active site of uPA and tPA. 27,32 The observed abrogation of recanalization by PAI-1 is in line with these previous reports, 32,33 and confirms the causative role of uPA and tPA in spontaneous recanalization of thromboembolized microvasculature. Our PAI-1 experiments further indicate that the heat shock-mediated upregulation of endogenous uPA and tPA does not only counteract the function of endogenous but also exogenously applied PAI-1.

The increase of endogenously expressed PAI-1 on heat shock induction was markedly less pronounced compared with the increase of uPA and tPA. This is in line with previous observations, demonstrating a stronger expression of uPA and tPA compared with PAI-1 in response to hyperthermia. 34 Although PAI-1 is a major stress-regulated gene, it is only weakly expressed in endothelial cells, 35 and may be further reduced in endothelial cells located close to fibrin clots. 34

The endogenous induction of a local pro-thrombolytic state by heat shock priming offers some advantages compared with the systemic application of plasminogen activators. Plasminogen activators are rapidly cleared from the systemic circulation, mainly by the function of the liver. 36 Additionally, the thrombolytic properties after local heat shock are restricted to the endangered microcirculation. Thus, systemic side effects, such as the assumed neurotoxicity of tPA, 37 adverse bleeding complications, 38 or anaphylactoid reactions related to recombinant tPA, 39 may be avoided.

Heat shock priming may represent a promising tool also in the clinical setting. Of interest, fever in septic conditions has been shown to induce heat shock proteins and to result in better respiratory function, lower blood lactate concentrations, and prolonged survival times. 40 These experimental results support clinical studies, demonstrating protective functions of intracellular heat shock protein-70 expression in patients with severe sepsis. 41 However, the effect of sepsis-associated fever on blood coagulation and thrombus formation remains to be determined.

In conclusion, we herein demonstrate that local heat shock priming induces endogenous hyperfibrinolysis by upregulation of plasminogen activators and consequently results in an accelerated and improved recanalization of thromboembolized microvasculature. Thus, the efficacy of local heat shock preconditioning should be further evaluated experimentally as a novel treatment strategy in disease states associated with microthromboembolization and infarction.

Acknowledgments

We appreciate the work of Klaus Saueressig and Janine Becker.

Source of Funding

This study was supported by the Deutsche Forschungsgemeinschaft (Me 900/1-3 and 1-4).

Disclosures

None.

【参考文献】
  Topol EJ, Yadav JS. Recognition of the importance of embolization in atherosclerotic vascular disease. Circulation. 2000; 101: 570-580.

Reimers B, Corvaja N, Moshiri S, Sacca S, Albiero R, Di Mario C, Pascotto P, Colombo A. Cerebral protection with filter devices during carotid artery stenting. Circulation. 2001; 104: 12-15.

Baim DS, Wahr D, George B, Leon MB, Greenberg J, Cutlip DE, Kaya U, Popma JJ, Ho KK, Kuntz RE. Randomized trial of a distal embolic protection device during percutaneous intervention of saphenous vein aorto-coronary bypass grafts. Circulation. 2002; 105: 1285-1290.

Borger MA, Ivanov J, Weisel RD, Rao V, Peniston CM. Stroke during coronary bypass surgery: principal role of cerebral macroemboli. Eur J Cardiothorac Surg. 2001; 19: 627-632.

Andresen DM, O?Shaughnessy M, Acland RD, Anderson GL, Schuscke D, Banis J Jr, Barker JH. Direct visualization and measurement of microsurgically induced thromboembolism. Microsurgery. 1994; 15: 413-420.

Sobel BE. Intracranial bleeding, fibrinolysis, and anticoagulation. Causal connections and clinical implications. Circulation. 1994; 90: 2147-2152.

De Maio A. The heat shock response. New Horizons. 1995; 3: 198-207.

Collen D, Lijnen HR. Basic and clinical aspects of fibrinolysis and thrombolysis. Blood. 1991; 78: 3114-3124.

Iba T, Shin T, Sonoda T, Rosales O, Sumpio BE. Stimulation of endothelial secretion of tissue-type plasminogen activator by repetitive stretch. J Surg Res. 1991; 50: 457-460.

Levin EG, Santell L, Salijooque F. Hyperosmotic stress stimulates tissue plasminogen activator expression by a PKC-independent pathway. Am J Physiol. 1993; 265: C387-C396.

Fukao H, Hagiya Y, Ueshima S, Okada K, Takaishi T, Izaki S, Matsuo O. Effect of heat shock on the expression of urokinase-type plasminogen activator receptor in human umbilical vein endothelial cells. Thromb Haemost. 1996; 75: 352-358.

Rücker M, Roesken F, Vollmar B, Menger MD. A novel approach for comparative study of periosteum, muscle, subcutis, and skin microcirculation by intravital fluorescence microscopy. Microvasc Res. 1998; 56: 30-42.

Overgaard K, Sereghy T, Boysen G, Pedersen H, Hoyer S, Diemer NH. A rat model reproducible cerebral infarction using thrombotic blood clot emboli. J Cereb Blood Flow Metab. 1992; 12: 484-490.

Loskutoff DJ, Samad F. The adipocyte and hemostatic balance in obesity studies of PAI-1. Arterioscler Thromb Vasc Biol. 1998; 18: 1-6.

Rücker M, Schäfer T, Stamm A, Saueressig K, Vollmar B, Spitzer WJ, Menger MD. New model for in vivo quantification of microvascular embolization, thrombus formation, and recanalization in composite flaps. J Surg Res. 2002; 108: 129-137.

Rücker M, Strobel O, Vollmar B, Roesken F, Menger MD. Vasomotion in critically perfused muscle protects adjacent tissues from capillary perfusion failure. Am J Physiol. 2000; 279: H550-H558.

Rücker M, Schäfer T, Roesken F, Spitzer WJ, Bauer M, Menger MD. Local heat shock priming-induced improvement in microvascular perfusion in osteomyocutaneous flaps is mediated by heat shock protein 32. Br J Surg. 2001; 88: 450-457.

Page C, Rose M, Yacoub M, Pigott R. Antigenic heterogeneity of vascular endothelium. Am J Pathol. 1992; 141: 673-683.

Derex L, Tomsick TA, Brott TG, Lewandowski CA, Frankel MR, Clark W, Starkman S, Spilker J, Udsten GJ, Khoury J, Grotta JC, Broderick JP. Outcome of stroke patients without angiographically revealed arterial occlusion within four hours of symptom onset. Am J Neuroradiol. 2001; 22: 685-690.

Kassem-Moussa H, Graffagnino C. Nonocclusion and spontaneous recanalization rates in acute ischemic stroke: a review of cerebral angiography studies. Arch Neurol. 2002; 59: 1870-1873.

Gargiulo NJ 3rd, Veith FJ, Lipsitz EC, Ohki T, Suggs WD, Cayne NS, Dadian N, Wain RA. Spontaneous recanalization of arterial occlusions: an unusual mechanism for symptomatic improvement. J Vasc Surg. 2002; 36: 1161-1166.

O?Shaughnessy M, Gu JM, Wyllie F, Acland RD, Anderson GL, Banis JC Jr, Barker JH. Microcirculatory consequences of microvascular surgery. Microsurgery. 1994; 15: 405-412.

Fuster V, Badimon L, Badimon JJ, Chesebro JH. The pathogenesis of coronary artery disease and the acute coronary syndromes. N Engl J Med. 1992; 326: 310-318.

Chandler AW. In vitro thrombotic coagulation of the blood. Lab Invest. 1958; 7: 110-114.

Krapohl BD, Siemionow M, Piza P, Pieramici SF, Zins JE. Thrombogenic stimulus causes long-term decrease of muscle flap perfusion. Ann Plast Surg. 1999; 42: 299-305.

Barnathan ES, Kuo A, Kariko K, Rosenfeld L, Murray SC, Behrendt N, Ronne E, Weiner D, Henkin J, Cines DB. Characterization of human endothelial cell urokinase-type plasminogen activator receptor protein and messenger RNA. Blood. 1990; 76: 1795-1806.

Emeis JJ. Perfused rat hindlegs. A model to study plasminogen activator release. Thromb Res. 1983; 30: 195-203.

Munkvad S. Fibrinolysis in patients with acute ischaemic heart disease. Dan Med Bull. 1993; 40: 383-408.

van Hinsbergh VWM, Sprengers ED, Kooistra T. Effect of thrombin on the production of plasminogen activators and PA-inhibitor-1 by human foreskin microvascular endothelial cells. Thromb Haemost. 1987; 57: 148-153.

Wojta J, Holzer M, Hufnagl P, Christ G, Hoover RL, Binder BR. Hyperthermia stimulates plasminogen activator inhibitor type 1 expression in human umbilical vein endothelial cells in vitro. Am J Pathol. 1991; 139: 911-919.

Ang C, Dawes J. The effects of hyperthermia on human endothelial monolayers: modulation of thrombotic potential and permeability. Blood Coagul Fibrinolysis. 1994; 5: 193-199.

Torr-Brown SR, Sobel BE. Attenuation of thrombolysis by release of plasminogen activator inhibitor type-1 from platelets. Thromb Res. 1993; 72: 413-421.

Farrehi PM, Ozaki CK, Carmeliet P, Fay WP. Regulation of arterial thrombolysis by plasminogen activator inhibitor-1 in mice. Circulation. 1998; 97: 1002-1008.

Fukao H, Matsuo O. Antithrombotic regulation in human endothelial cells by fibrinolytic factors. Semin Thromb Hemost. 2000; 26: 33-38.

Yamamoto K, Takeshita K, Shimokawa T, Yi H, Isobe K, Loskutoff DJ, Saito H. Plasminogen activator inhibitor-1 is a major stress-regulated gene: Implications for stress-induced thrombosis in aged individuals. PNAS. 2002; 99: 890-895.

Bounameaux H, Stassen JM, Seghers C, Collen D. Influence of fibrin and liver blood flow on the turnover and the systemic fibrinogenolytic effects of recombinant human tissue-type plasminogen activator in rabbits. Blood. 1986; 67: 1493-1497.

Vivien D, Buison A. Serine protease inhibitors: novel therapeutic targets for stroke? J Cereb Blood Flow Metab. 2000; 20: 755-764.

Nowak-Gottl U, Auberger K, Halimeh S, Junker R, Klinge J, Kreuz WD, Ries M, Schlegel N. Thrombolysis in newborns and infants. Thromb Haemost. 1999; 82: 112-116.

Hill MD, Barber PA, Takahashi J, Demchuk AM, Feasby TE, Buchan AM. Anaphylactoid reactions and angioedema during alteplase treatment of acute ischemic stroke. CMAJ. 2000; 162: 1281-1284.

Su F, Nguyen ND, Wang Z, Cai Y, Rogiers P, Vincent JL. Fever control in septic shock: beneficial or harmful? Shock. 2005; 23: 516-520.

Bruemmer-Smith S, Stuber F, Schroeder S. Protective functions of intracellular heat shock protein (HSP) 70-expression in patients with severe sepsis. Intensive Care Med. 2001; 27: 1835-1841.

作者单位：Institute for Clinical & Experimental Surgery (M.R., T.S., C.S., Y.H., M.M.), University of Saarland, Homburg/Saar; the Department of Oral and Maxillofacial Surgery (M.R.), Hannover Medical School, Hannover; and the Department of Experimental Surgery (B.V.), University of Rostock, Rostock, Germa