Blocking neutrophil diapedesis prevents hemorrhage during thrombocytopenia.

Spontaneous organ hemorrhage is the major complication in thrombocytopenia with a potential fatal outcome. However, the exact mechanisms regulating vascular integrity are still unknown. Here, we demonstrate that neutrophils recruited to inflammatory sites are the cellular culprits inducing thrombocytopenic tissue hemorrhage. Exposure of thrombocytopenic mice to UVB light provokes cutaneous petechial bleeding. This phenomenon is also observed in immune-thrombocytopenic patients when tested for UVB tolerance. Mechanistically, we show, analyzing several inflammatory models, that it is neutrophil diapedesis through the endothelial barrier that is responsible for the bleeding defect. First, bleeding is triggered by neutrophil-mediated mechanisms, which act downstream of capturing, adhesion, and crawling on the blood vessel wall and require Gαi signaling in neutrophils. Second, mutating Y731 in the cytoplasmic tail of VE-cadherin, known to selectively affect leukocyte diapedesis, but not the induction of vascular permeability, attenuates bleeding. Third, and in line with this, simply destabilizing endothelial junctions by histamine did not trigger bleeding. We conclude that specifically targeting neutrophil diapedesis through the endothelial barrier may represent a new therapeutic avenue to prevent fatal bleeding in immune-thrombocytopenic patients.

Blocking von Willebrand factor for treatment of cutaneous inflammation.

Von Willebrand factor (VWF), a key player in hemostasis, is increasingly recognized as a proinflammatory protein. Here, we found a massive accumulation of VWF in skin biopsies of patients suffering from immune complex (IC)-mediated vasculitis (ICV). To clarify the impact of VWF on cutaneous inflammation, we induced experimental ICV either in mice treated with VWF-blocking antibodies or in VWF(-/-) mice. Interference with VWF led to a significant inhibition of the cutaneous inflammatory response. We confirmed the major findings in irritative contact dermatitis, a second model of cutaneous inflammation. In vivo imaging of cutaneous inflammation in the dorsal skinfold chamber revealed unaffected leukocyte rolling on anti-VWF treatment. However, we identified that reduced leukocyte recruitment is accompanied by reduced vascular permeability. Although VWF-mediated neutrophil recruitment to the peritoneum was described to require the VWF receptor on platelets (glycoprotein Ibα (GPIbα)), the VWF/GPIbα axis was dispensable for cutaneous inflammation. As assessed in tail bleeding assays, we could exclude interference of VWF blockade with hemostasis. Of particular importance, anti-VWF treatment was effective both in prophylactic and therapeutic administration. Thus, VWF represents a promising target for the treatment of cutaneous inflammation, e.g., leukocytoclastic vasculitis.

New tools for assessing the individual risk of metastasis in renal cell carcinoma.

Localized renal cell carcinoma (RCC) progresses to metastatic disease in 20-40 % after surgical resection. Affected patients might benefit from adjuvant treatment and have to be reliably identified for treatment indication. However, existing molecular markers and classification nomograms lack sufficient validity for clinical application so far. Therefore, in order to improve diagnostic tools for the identification of patients at risk, we tested invasiveness and the capability to activate vascular endothelium of primary RCC cells as tumor specific functional parameters. As a parameter for cell invasiveness the ability of RCC cells to break-down transepithelial electrical resistance (TEER) of an epithelial cell monolayer was tested. Loss of resistance, calculated as invasivity index, resembled the degree of cell invasiveness. In addition, secretion of Von Willebrand Factor by endothelial cells incubated with RCC cell supernatant was measured as a surrogate marker for endothelial cell activation. TEER-assay results matched clinical status of disease in 9 out of 12 cases. Metastatic tumors and less differentiated tumors had a significant increase of invasivity index (p = 0.007; p = 0.034). Endothelial cell activation and clinical outcome matched in 5 out of 9 samples. In addition, tumor cell induced endothelial cell activation significantly correlated to the pathologic T classification status of RCC tumors (p = 0.009). Taken together, our study validated endothelial cell activation analysis and cell invasiveness as solitary prognostic markers for tumor dissemination. TEER-analysis has proven to be a useful functional assay giving highly relevant individual information on functional tumor cell characteristics that add to pathologic evaluation.

Human macrophage activation triggered by chitotriosidase-mediated chitin and chitosan degradation.

Chitin a biopolymer composed of N-acetyl-D-glucosamine (GlcNAc) residues is a structural component in human pathogens such as nematodes and fungi. Deacetylation of chitin generates chitosan which has been recently found in the cell wall of Cryptococcus neoformans a human pathogenic fungi causing life-threatening meningoencephalitis. While chitin and chitosan are currently studied as compounds of medical devices such as wound dressings or nanoparticles, its immunostimulatory potential and its metabolic fate in humans remains unclear. To gain more fundamental insights on the immunological properties of chitin and chitosan in humans we studied their degradation by chitotriosidase (ChT) and their inflammatory properties on human macrophages. Our data show that chitinhexamer is not able to increase the expression of inflammatory cytokines significantly. However, we measured an induction of ChT secretion upon chitinhexamer treatment. By analysis of human ChT-mediated cleavage of chitosan we could demonstrate a special mechanism of substrate cleavage, defined as processivity. Processivity enables the rapid production of small and diffusible chitin and chitosan fragments. In comparison to large chitosan polymers these ChT-produced small chitin/chitosan oligomers exhibit strongest macrophage activating properties reflected by an enhanced ChT secretion. Here we show that recognition of chitin and chitosan by human macrophages is triggered by the enzyme ChT due to the production of chitin and chitosan oligomers which in turn stimulates further ChT secretion and consequently oligomers production. Finally, we demonstrate that despite the high cooperativity of chitosan and chitin clearance by ChT seems very specific as no inflammatory response could be detected.

The mechanism of melanoma-associated thrombin activity and von Willebrand factor release from endothelial cells.

Activation of the coagulation system in malignancy enables tumor spreading and is thus associated with poor prognosis for the patient. In this study, we analyzed the in vitro mechanisms by which two human metastatic melanoma cell lines, MV3 and WM9, transform the vascular endothelium into a prothrombotic activated state. We show that both melanoma cell lines activate prothrombin due to tissue factor (TF) expression by showing that thrombin generation was blocked with a TF-neutralizing antibody and TF-siRNA. In addition, using the cysteine protease inhibitor E-64, we excluded the formerly described cancer procoagulant (CP) as a major factor contributing to thrombin generation. Furthermore, we describe a direct thrombin-independent response of endothelial cells (ECs) to MV3-derived supernatant as measured by rapid release of VWF. We also show that two clinically approved LMWHs, tinzaparin and enoxaparin, are effective inhibitors of thrombin generation and thrombin activity in plasma. Furthermore, our data indicate a protective effect of heparins on EC activation as shown by reduced VWF release in response to MV3 supernatant. These promising effects of heparins on the melanoma-induced thrombotic conditions justify further clinical investigations in the field of oncology.

Specific interaction between chitosan and matrix metalloprotease 2 decreases the invasive activity of human melanoma cells.

The crucial event in metastasis is tumor invasion which in the case of melanoma cells is dependent on matrix metalloprotease 2 (MMP2). Chitosan (MW ca. 5 x 10(5) g mol(-1), degree of acetylation ca. 30%) attenuated the invasive activity of melanoma cells in a cell-based invasion assay and reduced MMP2 activity in the supernatant of melanoma cells. While the expression level of MMP2 was not affected, the amount of MMP2 in the cell supernatant was reduced, indicating a posttranscriptional effect of chitosan on MMP2. Atomic force microscopy revealed a direct molecular interaction between MMP2 and chitosan forming a complex with a diameter of 349.0 +/- 69.06 nm and a height of 26.5 +/- 11.50 nm. Affinity chromatography revealed a high binding-specificity of MMP2 to chitosan, and a colorimetric MMP2 activity assay suggests a noncompetitive inhibition of MMP2 by chitosan. The possible use of chitosan as a new type of MMP2 inhibitor is discussed.

Tumor-derived matrix metalloproteinase-1 targets endothelial proteinase-activated receptor 1 promoting endothelial cell activation.

In the vascular system, circulating tumor cells interact with endothelial cells. Tumor-endothelial cross-talk transforms the intravascular milieu to a prothrombotic, proinflammatory, and cell-adhesive state called endothelial cell activation (ECA). In the present study, we analyze the potential of metastatic tumor-derived soluble factors to transform the vascular endothelium into a prothrombotic and proinflammatory activated state. Supernatant from cultured melanoma and colon cancer cells (A375, WM9, A7, and HT-29) induced an acute activation of macrovascular and microvascular endothelial cells (human umbilical vein endothelial cells and human dermal microvascular endothelial cells) as shown by intracellular calcium flux and secretion of von Willebrand factor and interleukin-8, all markers of acute ECA. This process was inhibited using specific proteinase-activated receptor 1 (PAR1) inhibitors (RWJ-58259 and SCH-79797), indicating a mediating role for endothelial thrombin receptors. Immunofluorescence, Western blot analysis, and collagenase activity assay of tumor cells and culture supernatant revealed the presence of matrix metalloproteinase-1 (MMP-1), a recently described activator of PAR1. Inhibition of MMP-1 in supernatant from cultured tumor cells significantly attenuated ECA. Additional studies using isolated human MMP-1 (5 nmol/L) proved the presence of a functional MMP-1/PAR1 axis in tumor-endothelial communication. These findings show a new pathway of tumor-endothelial cross-talk via an intravascular MMP1/PAR1 axis in microvascular and macrovascular endothelium. Inhibition of this cross-talk may be a powerful means to prevent tumor-induced ECA and thus thrombotic and inflammatory cell adhesion.

NF{kappa}B-dependent down-regulation of tumor necrosis factor receptor-associated proteins contributes to interleukin-1-mediated enhancement of ultraviolet B-induced apoptosis.

Activation of the transcription factor nuclear factor-kappaB (NFkappaB) by inflammatory cytokines like tumor necrosis (TNF) factor and interleukin-1 (IL-1) is generally associated with the induction of antiapoptotic pathways. Therefore, NFkappaB inhibits both intrinsically and extrinsically induced apoptosis and thus is regarded to act universally in an antiapoptotic fashion. Accordingly, activation of NFkappaB by IL-1 was shown to result in reduction of death ligand-induced apoptosis via up-regulation of antiapoptotic inhibitor of apoptosis proteins (IAPs). In contrast, apoptosis induced by ultraviolet-B radiation (UVB) was shown to be enhanced in an NFkappaB-dependent manner, indicating that NFkappaB can also act in a proapoptotic fashion. This study investigates the molecular mechanisms underlying IL-1-mediated enhancement of UVB-induced apoptosis. We show that NFkappaB activation in costimulation with UVB treatment results in repression of antiapoptotic genes and consequently in down-regulation of the respective proteins, like c-IAP, FLICE-inhibitory protein (FLIP), and some members of the TNF receptor-associated (TRAF)2 protein family. In parallel, TNFalpha is released, leading to activation of signaling pathways mediated by TNF receptor-1 (TNF-R1). Although TNF is well known to induce both proapoptotic and antiapoptotic effects, the down-regulated levels of TRAF-1, -2, and -6 proteins by IL-1 plus UVB action leads to a shift toward promotion of the proapoptotic pathway. In concert with the down-regulation of IAPs and FLIP, TNF-R1 activation as an additional proapoptotic stimulus now results in significant enhancement of UVB-induced apoptosis. Taken together, elucidation of the molecular mechanisms underlying IL-1-mediated enhancement of UVB-induced apoptosis revealed that NFkappaB does not exclusively act in an antiapoptotic fashion but may also mediate proapoptotic effects.

DNA damage, death receptor activation and reactive oxygen species contribute to ultraviolet radiation-induced apoptosis in an essential and independent way.

Nuclear DNA damage and death receptor (CD95) activation by ultraviolet-B radiation (UVB) play a major role in UVB-induced apoptosis. Removal of DNA damage combined with inhibition of death receptor activation resulted in pronounced but not complete suppression of apoptosis, indicating that a third independent pathway is involved. Since reactive oxygen species (ROS) cause apoptosis and are induced by UVB, the radical scavenger pyrrolidene-dithiocarbamate (PDTC) was used. PDTC prevented UVB-induced apoptosis partially, H(2)O(2)-induced cell death largely, but not CD95-mediated apoptosis. The same was observed for cytochrome c release from mitochondria, another important event during apoptosis. The proapoptotic protein Bid was cleaved upon exposure to UVB or to agonistic anti-CD95-antibodies, but not to H(2)O(2), indicating that H(2)O(2) uses a different pathway. The fact that PDTC neither inhibited CD95-mediated apoptosis nor affected UV-induced DNA damage indicated that ROS generated during UVB irradiation may directly trigger mitochondrial cytochrome c release, thereby contributing to apoptosis. Accordingly, complete inhibition of apoptosis was observed when in addition to DNA damage removal via photoreactivation and blockade of CD95 signaling by caspase-8 inhibitor zIETD, PDTC was added before UVB exposure. This indicates that DNA damage, death receptor activation and ROS formation contribute to UVB-induced apoptosis in an essential and independent way.