Matthias W. Laschke

Overcoming Intrinsic Multidrug Resistance in Melanoma by Blocking the Mitochondrial Respiratory Chain of Slow-Cycling JARID1Bhigh Cells

Despite success with BRAFV600E inhibitors, therapeutic responses in patients with metastatic melanoma are short-lived because of the acquisition of drug resistance. We identified a mechanism of intrinsic multidrug resistance based on the survival of a tumor cell subpopulation. Treatment with various drugs, including cisplatin and vemurafenib, uniformly leads to enrichment of slow-cycling, long-term tumor-maintaining melanoma cells expressing the H3K4-demethylase JARID1B/KDM5B/PLU-1. Proteome-profiling revealed an upregulation in enzymes of mitochondrial oxidative-ATP-synthesis (oxidative phosphorylation) in this subpopulation. Inhibition of mitochondrial respiration blocked the emergence of the JARID1B(high) subpopulation and sensitized melanoma cells to therapy, independent of their genotype. Our findings support a two-tiered approach combining anticancer agents that eliminate rapidly proliferating melanoma cells with inhibitors of the drug-resistant slow-cycling subpopulation.

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.

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.

Hydroxyethyl starch (130 kD), but not crystalloid volume support, improves microcirculation during normotensive endotoxemia.

Background Increased leukocyte–endothelial cell interaction (LE) and deterioration of capillary perfusion represent key mechanisms of septic organ dysfunction. The type of volume support, however, which may be used during septic disorders, remains controversial. Using intravital microscopy, the authors studied the effect of different regimens of clinically relevant volume support on endotoxin-induced microcirculatory disorders, including the synthetic colloid hydroxyethyl starch (HES, 130 kD) and a crystalloid regimen with isotonic saline solution (NaCl). Methods In Syrian Golden hamsters, normotensive endotoxemia was induced by intravenous application of Escherichia coli lipopolysaccharide (LPS, 2 mg/kg). The microcirculation was analyzed in striated muscle of skinfold preparations. HES 130 kD (Voluven®, 16 ml/kg, n = 7) or isotonic saline (NaCl, 66 ml/kg, n = 6) were infused 3 h after LPS exposure over a 1-h period (posttreatment mode). Animals receiving LPS without volume therapy served as control subjects (n = 8, control). LE, functional capillary density (FCD), and macromolecular leakage were repeatedly analyzed in the awake animals during a 24-h period using intravital fluorescence microscopy. Results HES 130 kD significantly reduced LPS-induced arteriolar and venular leukocyte adherence (P < 0.05), whereas NaCl resuscitation had no effect when compared with nontreated control animals. The LPS-induced decrease in FCD and increase in macromolecular leakage were also significantly attenuated by HES 130 kD but not by NaCl. Improvement of LPS-induced microcirculatory disorders by HES was unlikely the result of macro- and microhemodynamic changes because arterial blood pressure, heart rate, and venular wall shear rate did not differ between HES- and NaCl-treated animals. Conclusions Thus, our study provides microhemodynamic and cellular mechanisms of HES 130 kD-mediated protection on microcirculation during endotoxemia, even when used in a clinically relevant posttreatment mode during normotensive conditions.

In vitro and in vivo evaluation of a novel nanosize hydroxyapatite particles/poly(ester-urethane) composite scaffold for bone tissue engineering.

Scaffolds for bone tissue engineering should provide an osteoconductive surface to promote the ingrowth of new bone after implantation into bone defects. This may be achieved by hydroxyapatite loading of distinct scaffold biomaterials. Herein, we analyzed the in vitro and in vivo properties of a novel nanosize hydroxyapatite particles/poly(ester-urethane) (nHA/PU) composite scaffold which was prepared by a salt leaching-phase inverse process. Microtomography, scanning electron microscopy and X-ray spectroscopy analyses demonstrated the capability of the material processing to create a three-dimensional porous PU scaffold with nHA on the surface. Compared to nHA-free PU scaffolds (control), this modified scaffold type induced a significant increase in in vitro adsorption of model proteins. In vivo analysis of the inflammatory and angiogenic host tissue response to implanted nHA/PU scaffolds in the dorsal skinfold chamber model indicated that the incorporation of nHA particles into the scaffold material did not affect biocompatibility and vascularization when compared to control scaffolds. Thus, nHA/PU composite scaffolds represent a promising new type of scaffold for bone tissue engineering, combining the flexible material properties of PU with the advantage of an osteoconductive surface.

Platelet-dependent accumulation of leukocytes in sinusoids mediates hepatocellular damage in bile duct ligation-induced cholestasis

Although it is well known that extrahepatic cholestasis induces liver damage, the mechanisms are still not completely understood. The aim of the present study was to evaluate the role of platelets and P‐selectin in cholestasis‐induced liver injury.

Vasculogenesis: a new piece of the endometriosis puzzle

BACKGROUND; Endometriosis is a complex disease with a multifactorial pathogenesis, which is crucially dependent on the development of new blood vessels. Based on the current literature, the present review highlights the fact that the neovascularization of endometriotic lesions is not only driven by angiogenesis, but also involves de novo formation of microvessels from circulating endothelial progenitor cells (EPCs). This process, termed post-natal vasculogenesis, is a characteristic of various pathogenic conditions, such as tumour growth and atherosclerosis, and typically comprises the activation, mobilization and recruitment of bone marrow-derived EPCs to the sites of tissue hypoxia. METHODS ; Literature searches were performed in PubMed, MEDLINE and ISI Web of Knowledge for publications focusing on vasculogenesis in the endometrium and endometriotic lesions. RESULTS ; Recent studies indicate that up to 37% of the microvascular endothelium of ectopic endometrial tissue originates from EPCs, partly controlled by the stromal-cell-derived factor-1/chemokine receptor type 4 axis. Accordingly, blockade of EPC recruitment effectively inhibits the formation of microvascular networks in developing endometriotic lesions, indicating that vasculogenesis represents an integral part of the pathogenesis of endometriosis. CONCLUSIONS ; The involvement of vasculogenesis in endometriosis may offer the exciting opportunity for the future establishment of novel diagnostic and therapeutic strategies for this frequent gynaecological disease.

Rapamycin induces regression of endometriotic lesions by inhibiting neovascularization and cell proliferation

Rapamycin is a widely used drug with antifungal, immunosuppressant and antiangiogenic effects. Herein, we studied whether immunosuppressive doses of rapamycin are capable of influencing endometriotic lesions.

Protective effect of fasudil, a Rho-kinase inhibitor, on chemokine expression, leukocyte recruitment, and hepatocellular apoptosis in septic liver injury

Rho‐kinase signaling regulates important features of inflammatory reactions. Herein, we investigated the effect and mechanisms of action of the Rho‐kinase inhibitor fasudil in endotoxemic liver injury. C57/BL/6 mice were challenged with lipopolysaccharide (LPS) and D‐galactosamine, with or without pretreatment with the Rho‐kinase inhibitor fasudil. Six hours after endotoxin challenge, leukocyte‐endothelium interactions in the hepatic microvasculature were studied by use of intravital fluorescence microscopy and tumor necrosis factor α (TNF‐α); CXC chemokines as well as liver enzymes and apoptosis were determined. Administration of fasudil reduced LPS‐induced leukocyte adhesion in postsinusoidal venules and sequestration in sinusoids. Moreover, we found that fasudil abolished extravascular infiltration of leukocytes as well as production of TNF‐α and CXC chemokines in the liver of endotoxemic mice. Liver enzymes and hepatocellular apoptosis were markedly reduced, and sinusoidal perfusion was improved significantly in endotoxemic mice pretreated with fasudil. Our novel data document that fasudil is a potent inhibitor of endotoxin‐induced expression of TNF‐α and CXC chemokines as well as leukocyte infiltration and hepatocellular apoptosis in the liver. Based on the present findings, it is suggested that inhibition of the Rho‐kinase signaling pathway may be a useful target in the treatment of septic liver injury.

Improvement of Vascularization of Plga Scaffolds by Inosculation of In Situ-preformed Functional Blood Vessels With the Host Microvasculature

Objective:We analyzed, in vivo, whether the establishment of blood supply to implanted scaffolds can be accelerated by inosculation of an in situ-preformed microvascular network with the host microvasculature. Background:A rapid vascularization is crucial for the survival of scaffold-based transplanted tissue constructs. Methods:Poly-lactic-glycolic acid scaffolds were implanted into the flank of balb/c or green fluorescent protein (GFP)-transgenic mice for 20 days to create in situ a new microvascular network within the scaffolds. The prevascularized scaffolds were then transferred into the dorsal skinfold chamber of isogeneic recipient mice. Nonvascularized poly-lactic-glycolic acid scaffolds served as controls. Vascularization, blood perfusion, and cell survival of the implants were analyzed over 14 days using intravital fluorescence microscopy, histology, and immunohistochemistry. Results:Our results demonstrate that establishment of blood perfusion of prevascularized scaffolds is significantly accelerated and improved (136.7 ± 23.2 pl/s) when compared with controls (6.9 ± 1.9 pl/s), because the in situ-preformed microvessels were reperfused by forming interconnections to the host microvasculature. Apoptotic cell death within the implants was found only during the first 3 to 6 days after scaffold implantation during lack of blood perfusion, but not during the further 14-day observation period. Conclusions:Inosculation of in situ-preformed functional blood vessels represents a promising approach to improve the blood supply to implanted tissue constructs.