Michael D. Menger

Combined inhibition of VEGF and PDGF signaling enforces tumor vessel regression by interfering with pericyte-mediated endothelial cell survival mechanisms

Destruction of existing tumor blood vessels may be achieved by targeting vascular endothelial growth factor (VEGF) signaling, which mediates not only endothelial cell proliferation but also endothelial cell survival. In this study, however, intravital microscopy failed to demonstrate that targeting of VEGFR‐2 (by the tyrosine kinase inhibitor SU5416) induces significant regression of experimental tumor blood vessels. Immunohistochemistry, electron microscopy, expression analyses, and in situ hybridization provide evidence that this resistance of tumor blood vessels to VEGFR‐2 targeting is conferred by pericytes that stabilize blood vessels and provide endothelial cell survival signals via the Ang‐1/Tie2 pathway. In contrast, targeting VEGFR‐2 plus the plate‐let‐derived growth factor receptor (PDGFR)‐β system (PDGFR‐β signaling (by SU6668) rapidly forced 40% of tumor blood vessels into regression, rendering these tumors hypoxic as shown by phosphorescence quenching. TUNEL staining, electron microscopy, and apoptosis blocking experiments suggest that VEGFR‐2 plus PDGFR‐β targeting enforced tumor blood vessel regression by inducing endothelial cell apoptosis. We further show that this is achieved by an interference with pericyte‐endothelial cell interaction. This study provides novel insights into the mechanisms of how 1) pericytes may provide escape strategies to anti‐angiogenic therapies and 2) novel concepts that target not only endothelial cells but also pericyte‐associated pathways involved in vascular stabilization and maturation exert potent anti‐vascular effects.

The Hepatic Microcirculation: Mechanistic Contributions and Therapeutic Targets in Liver Injury and Repair

The complex functions of the liver in biosynthesis, metabolism, clearance, and host defense are tightly dependent on an adequate microcirculation. To guarantee hepatic homeostasis, this requires not only a sufficient nutritive perfusion and oxygen supply, but also a balanced vasomotor control and an appropriate cell-cell communication. Deteriorations of the hepatic homeostasis, as observed in ischemia/reperfusion, cold preservation and transplantation, septic organ failure, and hepatic resection-induced hyperperfusion, are associated with a high morbidity and mortality. During the last two decades, experimental studies have demonstrated that microcirculatory disorders are determinants for organ failure in these disease states. Disorders include 1) a dysregulation of the vasomotor control with a deterioration of the endothelin-nitric oxide balance, an arterial and sinusoidal constriction, and a shutdown of the microcirculation as well as 2) an overwhelming inflammatory response with microvascular leukocyte accumulation, platelet adherence, and Kupffer cell activation. Within the sequelae of events, proinflammatory mediators, such as reactive oxygen species and tumor necrosis factor-alpha, are the key players, causing the microvascular dysfunction and perfusion failure. This review covers the morphological and functional characterization of the hepatic microcirculation, the mechanistic contributions in surgical disease states, and the therapeutic targets to attenuate tissue injury and organ dysfunction. It also indicates future directions to translate the knowledge achieved from experimental studies into clinical practice. By this, the use of the recently introduced techniques to monitor the hepatic microcirculation in humans, such as near-infrared spectroscopy or orthogonal polarized spectral imaging, may allow an early initiation of treatment, which should benefit the final outcome of these critically ill patients.

Liver Ischemia And Reperfusion Induces A Systemic Inflammatory Response Through Kupffer Cell Activation

To study the role of Kupffer cells (KC) as a cellular source of proinflammatory cytokines in hepatic ischemia/reperfusion, Sprague-Dawley rats were subjected to 20 min global hepatic ischemia. Sham-operated animals served as controls. Blood levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 alpha (IL-1 alpha), and interleukin 6 (IL-6) were determined after 10, 30, 60, 120, and 240 min of reperfusion and compared with spontaneous cytokine release by KC isolated after 60 min of reperfusion. Hepatic ischemia/reperfusion resulted in an enhanced (p < .01) spontaneous release of TNF-alpha (+482%), IL-1 alpha (+33%), and IL-6 (+175%) by KC. Kinetic analysis of cytokinemia revealed an early increase (p < .01) of TNF-alpha and IL-1 alpha within minutes upon reperfusion, while an elevation of IL-6 serum levels was observed with a delay of 2 h. Early cytokinemia was associated with dysfunction/injury of the liver, lung, and kidney after 4 and 24 h of reperfusion, respectively. These data indicate that hepatic ischemia/reperfusion results in Kupffer cell activation and increased cytokine levels, which may produce systemic inflammation and may be responsible for tissue injury locally and on remote sites.

Microtumor growth initiates angiogenic sprouting with simultaneous expression of VEGF, VEGF receptor-2, and angiopoietin-2

Tumors have been thought to initiate as avascular aggregates of malignant cells that only later induce vascularization. Recently, this classic concept of tumor angiogenesis has been challenged by the suggestion that tumor cells grow by co-opting preexisting host vessels and thus initiate as well-vascularized tumors without triggering angiogenesis. To discriminate between these two mechanisms, we have used intravital epifluorescence microscopy and multi-photon laser scanning confocal microscopy to visualize C6 microglioma vascularization and tumor cell behavior. To address the mechanisms underlying tumor initiation, we assessed the expression of VEGF, VEGF receptor-2 (VEGFR-2), and angiopoietin-2 (Ang-2), as well as endothelial cell proliferation. We show that multicellular aggregates (<< 1 mm(3)) initiate vascular growth by angiogenic sprouting via the simultaneous expression of VEGFR-2 and Ang-2 by host and tumor endothelium. Host blood vessels are not co-opted by tumor cells but rather are used as trails for tumor cell invasion of the host tissue. Our data further suggest that the established microvasculature of growing tumors is characterized by a continuous vascular remodeling, putatively mediated by the expression of VEGF and Ang-2. The results of this study suggest a new concept of vascular tumor initiation that may have important implications for the clinical application of antiangiogenic strategies.

Enhanced repair of articular cartilage defects in vivo by transplanted chondrocytes overexpressing insulin-like growth factor I (IGF-I)

Traumatic articular cartilage lesions have a limited capacity to heal. We tested the hypothesis that overexpression of a human insulin-like growth factor I (IGF-I) cDNA by transplanted articular chondrocytes enhances the repair of full-thickness (osteochondral) cartilage defects in vivo. Lapine articular chondrocytes were transfected with expression plasmid vectors containing the cDNA for the Escherichia coli lacZ gene or the human IGF-I gene and were encapsulated in alginate. The expression patterns of the transgenes in these implants were monitored in vitro for 36 days. Transfected allogeneic chondrocytes in alginate were transplanted into osteochondral defects in the trochlear groove of rabbits. At three and 14 weeks, the quality of articular cartilage repair was evaluated qualitatively and quantitatively. In vitro, IGF-I secretion by implants constructed from IGF-I-transfected chondrocytes and alginate was 123.2±22.3 ng/107 cells/24 h at day 4 post transfection and remained elevated at day 36, the longest time point evaluated. In vivo, transplantation of IGF-I implants improved articular cartilage repair and accelerated the formation of the subchondral bone at both time points compared to lacZ implants. The data indicate that allogeneic chondrocytes, transfected by a nonviral method and cultured in alginate, are able to secrete biologically relevant amounts of IGF-I over a prolonged period of time in vitro. The data further demonstrate that implantation of these composites into deep articular cartilage defects is sufficient to augment cartilage defect repair in vivo. These results suggest that therapeutic growth factor gene delivery using encapsulated and transplanted genetically modified chondrocytes may be applicable to sites of focal articular cartilage damage.

Leukocytes contribute to hepatic ischemia/reperfusion injury via intercellular adhesion molecule-1-mediated venular adherence*

BACKGROUND Leukocytes are suggested to modulate ischemia/reperfusion injury via membrane receptor-controlled interaction with the microvascular endothelium. METHODS With the use of intravital fluorescence microscopy we investigated the role of the intercellular adhesion molecule-1 (ICAM-1) in a rat model of hepatic reperfusion injury with a neutralizing monoclonal antibody (anti-ICAM-1). RESULTS Sixty minutes of left lobar ischemia and reperfusion (isotype-matched immunoglobulin G1 control antibody) caused leukostasis in sinusoids (240 +/- 15 cells per liver lobule), leukocyte adherence in postsinusoidal venules (679 +/- 76 cells per mm2 endothelial surface of postsinusoidal venules), nutritive perfusion failure (15% +/- 2% nonperfused sinusoids), excretory dysfunction (bile flow, 1.2 +/- 0.3 microliters.min-1.gm-1), and loss of hepatocellular integrity (serum aspartate aminotransferase, 1353 +/- 317 units.L-1; serum alanine aminotransferase, 1055 +/- 265 units.L-1). Anti-ICAM-1 did not affect sinusoidal leukostasis; however, it effectively inhibited postischemic leukocyte adherence to the venular endothelial lining (217 +/- 38 cells/mm2, p < 0.01). Concomitantly, hepatic reperfusion injury, including sinusoidal perfusion (6% +/- 1% nonperfused sinusoids, p < 0.01), excretory function (bile flow, 1.8 +/- 0.1 microliters.min-1.gm-1, p < 0.05), and hepatocellular integrity (aspartate aminotransferase, 480 +/- 108 units.L-1; alanine aminotransferase, 447 +/- 80 units.L-1, p < 0.05), was significantly ameliorated by anti-ICAM-1. CONCLUSIONS These findings prove in vivo the pivotal role of ICAM-1 in leukocyte-dependent manifestation of postischemic liver damage.

Surgical trauma: hyperinflammation versus immunosuppression?

BackgroundExperimental and clinical studies have brought evidence that surgical trauma markedly affects the immune system, including both the specific and the non-specific immune response.Materials and methodsThis report reviews the present knowledge on the mechanisms of surgical trauma-induced immune dysfunction and outlines experimental and clinical approaches to find effective treatment strategies.ResultsMajor surgical trauma induces an early hyperinflammatory response, which is characterized by (1) pro-inflammatory tumour necrosis factor alpha (TNF), interleukin (IL)-1, and IL-6 cytokine release and (2) neutrophil activation and microvascular adherence, as well as (3) uncontrolled polymorphonuclear (PMN) and macrophage oxidative burst. The massive and continuous IL-6 release induces an acute phase response, but, more importantly, also accounts for the up-regulation of major anti-inflammatory mediators, such as prostaglandin (PG) E2, IL-10 and transforming growth factor (TGF)-ß. This results in surgical, trauma-induced, immunosuppression, as indicated by (1) monocyte deactivation, reflected by the lack of monocytic TNF- production upon lipopolysaccharide (LPS) stimulation, and (2) a shift of the Th1/Th2 ratio towards a Th2-dominated cytokine pattern. The imbalance between pro-inflammatory and anti-inflammatory cytokines and immuno-competent cells determines the phenotype of disease and should help the physician to compose the therapeutic strategy. In fact, recent clinical studies have shown that both the initial uncontrolled hyperinflammation and the continued cell-mediated immunosuppression represent primary targets to counteract post-surgery immune dysfunction. The balance between inflammatory and anti-inflammatory forces may be restored by interferon gamma (IFN-γ) to counteract monocyte deactivation; the anti-inflammatory PGE2 may be inhibited by indomethacin to attenuate immunosuppression; or the initial hyperinflammation may be targeted by administration of anti-inflammatory substances, such as granulocyte colony-stimulating factor (G-CSF), hydoxyethyl starch, or pentoxifylline.ConclusionsWhen drawing up the therapeutic regimen the physician should not consider hyperinflammation versus immunosuppression, but hyperinflammation and immunosuppression, aiming at restoring an appropriate mediator- and immune cell-associated balance.

Microhemodynamic and cellular mechanisms of activated protein C action during endotoxemia.

ObjectiveTo characterize microcirculatory actions of activated protein C in an endotoxemia rodent model that allows in vivo studies of microvascular inflammation and perfusion dysfunction. DesignAnimal study using intravital microscopy. SettingAnimal research facility. SubjectsMale Syrian golden hamsters, 6–8 wks old with a body weight of 60–80 g. InterventionsIn skinfold preparations, endotoxemia was induced by intravenous administration of 2 mg/kg endotoxin (lipopolysaccharide, Escherichia coli). Intravital microscopy allowed quantitative analysis of arteriolar and venular leukocyte adhesion and functional capillary density (cm−1) that served as a measure of microvascular perfusion failure. Activated protein C (APC group, n = 8, 24 &mgr;g/kg intravenously) was substituted continuously during 8 hrs after lipopolysaccharide, whereas endotoxemic buffer-treated animals (control, n = 7) served as controls. Measurements and Main ResultsLipopolysaccharide increased leukocyte adhesion and decreased functional capillary density to 50% of baseline values (p < .01 vs. baseline). Activated protein C treatment inhibited (p < .05) lipopolysaccharide-mediated leukocytic response and attenuated (p < .05) endotoxic perfusion failure in nutritive capillaries. ConclusionsActivated protein C-induced protection from lipopolysaccharide-mediated microcirculatory dysfunction was characterized in vivo for the first time. The impressive modification of leukocyte cross-talk indicates systemic anti-inflammatory activated protein C effects on leukocytes and the endothelium, subsequently improving capillary perfusion. These actions could represent the in vivo mechanism of activated protein C interactions observed in patients with severe sepsis.

Comparison of surgical stress between laparoscopic and open colonic resections

Background: The magnitude of surgical trauma after laparoscopic and open colonic resection was evaluated by examining postoperative serum values of interleukin-6 (IL-6), IL-10, C-reactive protein (CRP), and granulocyte elastase (GE) for further evidence of the benefit realized with minimally invasive approaches in colonic surgery. Methods: Altogether, 42 patients with Crohns disease (n = 20) or colon carcinomas/adenomas (n = 22) were matched by age, gender, body mass index (BMI), and Crohns Disease Activity Index for either a laparoscopic (n = 21) or an open colonic resection (n = 21). In both groups the postoperative serum levels of IL-6, IL-10, C-RP, and granulocyte elastase were determined, as indicators of surgical stress. Results: Laparoscopic and open colonic resection caused a significant increase in serum IL-6, IL-10, CRP, and granulocyle elastase levels. The comparison between laparoscopic and open colonic resections, however, showed significantly lower serum IL-6, IL-10, CRP, and granulocyte elastase levels after laparoscopic colonic resection, which was most evident for IL-6 and granulocyte elastase. Conclusions: Our study demonstrated that IL-6 and granulocyte elastase may be appropriated particularly to monitor surgical stress. By using these parameters, we found a significant reduction in surgical trauma after laparoscopic surgery, was compared with the open procedure. This supports the clinical findings of a clear benefit for patients undergoing laparoscopic colonic surgery.

Viewing the Microcirculation through the Window: Some Twenty Years Experience with the Hamster Dorsal Skinfold Chamber

Intravital microscopy represents a sophisticated technique to study the microcirculation in health and disease. While most preparations used for those studies are acute in nature, the use of chamber preparations in the skinfold bear the advantage to allow for chronic studies with repeated analysis of the microcirculation over a prolonged period of time. The skinfold chamber model for microcirculatory analysis has been adapted to mice, rats and hamsters. Although the use of rats and, in particular, the use of mice has the advantage of the availability of species-specific tools, the use of the hamster as the experimental animal may be preferred due to anatomical reasons, which facilitate the microsurgical preparation and improve the quality of microscopic imaging. The use of the hamster dorsal skinfold chamber, firstly described by Endrich and coworkers in 1980, has brought out during the last two decades a considerable number of experimental studies within the fields of microcirculation physiology, inflammation and sepsis, ischemia-reperfusion, angiogenesis, and transplantation, indicating that the model has to be considered a versatile tool to study the microcirculation in health and disease.