Cellular stress induces erythrocyte assembly on intravascular von Willebrand factor strings and promotes microangiopathy.

Microangiopathy with subsequent organ damage represents a major complication in several diseases. The mechanisms leading to microvascular occlusion include von Willebrand factor (VWF), notably the formation of ultra-large von Willebrand factor fibers (ULVWFs) and platelet aggregation. To date, the contribution of erythrocytes to vascular occlusion is incompletely clarified. We investigated the platelet-independent interaction between stressed erythrocytes and ULVWFs and its consequences for microcirculation and organ function under dynamic conditions. In response to shear stress, erythrocytes interacted strongly with VWF to initiate the formation of ULVWF/erythrocyte aggregates via the binding of Annexin V to the VWF A1 domain. VWF-erythrocyte adhesion was attenuated by heparin and the VWF-specific protease ADAMTS13. In an in vivo model of renal ischemia/reperfusion injury, erythrocytes adhered to capillaries of wild-type but not VWF-deficient mice and later resulted in less renal damage. In vivo imaging in mice confirmed the adhesion of stressed erythrocytes to the vessel wall. Moreover, enhanced eryptosis rates and increased VWF binding were detected in blood samples from patients with chronic renal failure. Our study demonstrates that stressed erythrocytes have a pronounced binding affinity to ULVWFs. The discovered mechanisms suggest that erythrocytes are essential for the pathogenesis of microangiopathies and renal damage by actively binding to ULVWFs.

Endothelial glycocalyx breakdown is mediated by angiopoietin-2.

AIMS: The endothelial glycocalyx (eGC), a carbohydrate-rich layer lining the luminal surface of the endothelium, provides a first vasoprotective barrier against vascular leakage and adhesion in sepsis and vessel inflammation. Angiopoietin-2 (Angpt-2), an antagonist of the endothelium-stabilizing receptor Tie2 secreted by endothelial cells, promotes vascular permeability through cellular contraction and junctional disintegration. We hypothesized that Angpt-2 might also mediate the breakdown of the eGC. METHODS AND RESULTS: Using confocal and atomic force microscopy, we show that exogenous Angpt-2 induces a rapid loss of the eGC in endothelial cells in vitro. Glycocalyx deterioration involves the specific loss of its main constituent heparan sulphate, paralleled by the secretion of the heparan sulphate-specific heparanase from late endosomal/lysosomal stores. Corresponding in vivo experiments revealed that exogenous Angpt-2 leads to heparanase-dependent eGC breakdown, which contributes to plasma leakage and leukocyte recruitment in vivo. CONCLUSION: Our data indicate that eGC breakdown is mediated by Angpt-2 in a non-redundant manner.

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.

A Phase II Multicenter Trial With Rivaroxaban in the Treatment of Livedoid Vasculopathy Assessing Pain on a Visual Analog Scale.

BACKGROUND: Livedoid vasculopathy is an orphan skin disease characterized by recurrent thrombosis of the cutaneous microcirculation. It manifests itself almost exclusively in the ankles, the back of the feet, and the distal part of the lower legs. Because of the vascular occlusion, patients suffer from intense local ischemic pain. Incidence of livedoid vasculopathy is estimated to be around 1:100,000. There are currently no approved treatments for livedoid vasculopathy, making off-label therapy the only option. In Europe, thromboprophylactic treatment with low-molecular-weight heparins has become widely accepted. OBJECTIVE: The aim of this trial is the statistical verification of the therapeutic effects of the anticoagulant rivaroxaban in patients suffering from livedoid vasculopathy. METHODS: We performed a therapeutic phase IIa trial designed as a prospective, one-armed, multicenter, interventional series of cases with a calculated sample size of 20 patients. The primary outcome is the assessment of local pain on the visual analog scale (VAS) as an intraindividual difference of 2 values between baseline and 12 weeks. RESULTS: Enrollment started in December 2012 and was still open at the date of submission. The study is expected to finish in November 2014. CONCLUSIONS: Livedoid vasculopathy is associated with increased thrombophilia in the cutaneous microcirculation and the continuous use of anticoagulants helps improve the symptoms. The causes of cutaneous infarctions are heterogenous, but ultimately follow the known mechanisms of the coagulation cascade. Rivaroxaban affects the coagulation cascade and inhibits the factor Xa-dependent conversion of prothrombin to thrombin, thereby considerably reducing the risk of thrombosis. TRIAL REGISTRATION: Trial Registration EudraCT Number: 2012-000108-13-DE; https://www.clinicaltrialsregister.eu/ctr-search/search?query=eudract_number:2012-000108-13 (Archived by WebCite at http://www.webcitation.org/6UCktWVCA); German Clinical Trials Register (DRKS): DRKS00004652; https://drks-neu.uniklinik-freiburg.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00004652 (Archived by WebCite at http://www.webcitation.org/6UCIAKyCS).

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.

α-1-Antitrypsin and IFN-γ reduce the severity of IC-mediated vasculitis by regulation of leukocyte recruitment in vivo.

IC-mediated vasculitis (ICV) can be life threatening. The cellular and immune mechanisms controlling ICV are poorly understood. Therefore, we investigated the role of α-1-antitrypsin (α1AT) and IFN-γ in reducing the severity of ICV in a mouse model in vivo. To induce ICV, mice were challenged with the reverse passive Arthus reaction (RPA), the prototypic in vivo model for leukocytoclastic vasculitis (LcV), and the modulation of vascular permeability, edema formation, and leukocyte recruitment was studied. To further analyze the dynamics of RPA, we applied intravital microscopy in the dorsal skinfold chamber. α1AT continuously led to reduced leukocyte recruitment. α1AT interfered with neutrophil recruitment through a KC-dependent mechanism and reduced KC-elicited neutrophil activation. In contrast to α1AT, IFN-γ-reduced leukocyte recruitment during RPA was clearly independent of KC. We also revealed that the recruitment of neutrophils during RPA was a prerequisite for full KC expression. Thus, therapeutic administration of α1AT and IFN-γ might be beneficial for limiting the duration and severity of ICV.

Anti-inflammatory effects of FTY720 do not prevent neuronal cell loss in a rat model of optic neuritis.

In multiple sclerosis, long-term disability is caused by axonal and neuronal damage. Established therapies target primarily the inflammatory component of the disease, but fail to prevent neurodegeneration. Fingolimod (codenamed FTY720) is an oral sphingosine 1-phosphate (S1P) receptor modulator with promising results in phase II trials in multiple sclerosis patients and is under further development as a novel treatment for multiple sclerosis. To evaluate whether FTY720 has neuroprotective properties, we tested this drug in a rat model of myelin oligodendrocyte glycoprotein-induced optic neuritis. FTY720 exerted significant anti-inflammatory effects during optic neuritis and reduced inflammation, demyelination, and axonal damage; however, FTY720 treatment did not prevent apoptosis of retinal ganglion cells (RGCs), the neurons that form the axons of the optic nerve. Consistent with this lack of effect on RGC survival, FTY720 treatment did not improve visual function, nor did it prevent apoptosis of RGCs in vitro. We observed a persistent activation of apoptotic signaling pathways in RGCs under FTY720 treatment, a possible underlying mechanism for the lack of neuroprotection in the presence of strong anti-inflammatory effects, Furthermore, FTY720 shifted the remaining inflammation in the optic nerve toward neurotoxicity by modest up-regulation of potential neurotoxic cytokines. We conclude that FTY720-induced anti-inflammation and axon protection did not of itself protect neurons from apoptotic cell death.