Blood-brain barrier (BBB) disruption, resulting from loss of tight junctions (TJ) and activation of matrix metalloproteinases (MMPs), is associated with edema formation in ischemic stroke. Cerebral edema develops in a phasic manner and consists of both vasogenic and cytotoxic components. Although it is contingent on several independent mechanisms, involving hypoxic and inflammatory responses, the single effect of prolonged hypoxia on BBB integrity in vivo was not addressed so far. Exposing mice to normobaric hypoxia (8% oxygen for 48 h) led to a significant increase in vascular permeability associated with diminished expression of the TJ protein occludin. Immunofluorescence studies revealed that hypoxia resulted in disrupted continuity of occludin and zonula occludens-1 (Zo-1) staining with significant gap formation. Hypoxia increased gelatinolytic activity specifically in vascular structures and gel zymography identified MMP-9 as enzymatic source. Treatment with an MMP inhibitor reduced vascular leakage and attenuated disorganization of TJ. Inhibition of vascular endothelial growth factor (VEGF) attenuated vascular leakage and MMP-9 activation induced by hypoxia. In conclusion, our data suggest that hypoxia-induced edema formation is mediated by MMP-9-dependent TJ rearrangement by a mechanism involving VEGF. Therefore, inhibition of MMP-9 might provide the basis for therapeutic strategies to treat brain edema.

Key Points• Tumor-derived VEGF-A mediates endothelial cell activation, VWF release, and platelet aggregation provoking coagulation in tumor patients.• Local ADAMTS13 inhibition promotes VWF fiber formation in tumor microvessels.Tumor-mediated procoagulatory activity leads to venous thromboembolism and supports metastasis in cancer patients. A prerequisite for metastasis formation is the interaction of cancer cells with endothelial cells (ECs) followed by their extravasation. Although it is known that activation of ECs and the release of the procoagulatory protein von Willebrand factor (VWF) is essential for malignancy, the underlying mechanisms remain poorly understood. We hypothesized that VWF fibers in tumor vessels promote tumor-associated thromboembolism and metastasis. Using in vitro settings, mouse models, and human tumor samples, we showed that melanoma cells activate ECs followed by the luminal release of VWF fibers and platelet aggregation in tumor microvessels. Analysis of human blood samples and tumor tissue revealed that a promoted VWF release combined with a local inhibition of proteolytic activity and protein expression of ADAMTS13 (a disintegrinlike and metalloproteinase with thrombospondin type I repeats 13) accounts for this procoagulatory milieu. Blocking endothelial cell activation by the low-molecular-weight heparin tinzaparin was accompanied by a lack of VWF networks and inhibited tumor progression in a transgenic mouse model. Our findings implicate a mechanism wherein tumor-derived vascular endothelial growth factor-A (VEGF-A) promotes tumor progression and angiogenesis. Thus, targeting EC activation envisions new therapeutic strategies attenuating tumor-related angiogenesis and coagulation. (Blood. 2015;125(20):3153-3163) IntroductionTo form new metastatic lesions, circulating melanoma cells have to interact with endothelial cells (ECs) and migrate through the vessel wall.1,2 In this context, our own in vitro studies show that melanoma cells activate ECs by an indirect, tissue factor (TF)-mediated thrombin generation.3 Next to this indirect melanoma-induced EC activation, recent findings identified melanoma-derived vascular endothelial growth factor-A (VEGF-A) as main mediator of direct EC activation. 4,5 Both the thrombin-and the VEGF-A-dependent pathways induce EC activation followed by Weibel-Palade body (WPB) exocytosis and the release of inflammatory cytokines and the highly procoagulatory glycoprotein von Willebrand factor (VWF), linking inflammation and coagulation. 6 On the one hand, luminally released VWF fibers are involved in hemostasis and vessel repair as mediators of platelet adhesion to the endothelium. 7,8 On the other hand, we showed that tumor cell-induced ultra-large VWF (ULVWF) fibers have the highest potential for platelet binding and aggregation.9,10 This effect may contribute not only to pathophysiologic vessel occlusion, 11 but also to the establishment of metastasis as platelets facilitate tumor cell extravasation. 12-14Indeed, it is well-known that cancer pati...

Abstract-The evolutionary conserved Wnt signaling pathway regulates cardiogenesis. However, members of the Wnt pathway are also expressed in the adult heart. Although Wnt-signaling is quiescent under normal conditions, we noticed activation on pathological stress of the heart, such as chronic afterload increase. To examine the role of Wnt signaling on the postnatal heart, we modified the expression and function of the Wnt regulator dishevelled 1 (Dvl-1) both in transgenic mice with cardiac-specific overexpression of Dvl-1 (Dvl-1-Tg) and in cultured cardiac myocytes. Dvl-1-Tg mice (3 months) had severe cardiac hypertrophy (heart weight:body weight ratio: 5.2Ϯ0.3 mg/g wild-type [WT] versus 6.4Ϯ0.7 mg/g Dvl-1-Tg; PϽ0.01), an increase in cardiomyocyte size (86% increase in Dvl-1-Tg compared with WT; PϽ0.01) and marked raise of atrial natriuretic factor expression (12-fold increase versus WT; PϽ0.01). Hypertrophy was associated with left ventricular dilatation in Dvl-1-Tg and a reduction of ejection fraction (4.4Ϯ0.1 mm versus 5.5Ϯ0.2 mm, 80Ϯ2% and 43Ϯ4% in WT versus Dvl-1-Tg, respectively; PϽ0.01). Transgenic animals died prematurely before 6 months of age. Both canonical as well as noncanonical Wnt signaling branches were activated in the Dvl-1-Tg animals. Small interfering RNA-mediated depletion of Dvl-1 was used to further characterize the role of Dvl-1 in cardiac myocytes. Whereas baseline parameters were unaltered, ␤-adrenergic hypertrophic response was abrogated in Dvl-1 knockdown cardiac myocytes, indicating a mandatory role in ␤-adrenergic stimulation. Therefore, activation of Wnt signaling is sufficient and critical for the induction of myocardial hypertrophy and cardiomyopathy. Key Words: Wnt signaling Ⅲ cardiac hypertrophy Ⅲ experimental heart failure A fter conditions of increased wall stress, the myocardium may adopt by a process called remodeling. Left ventricular remodeling is considered to be a maladaptive process characterized by myocyte hypertrophy, an increase in myocardial fibrosis, and left ventricular dilatation. Remodeling may induce clinically overt heart failure and contributes to increased mortality after myocardial infarction. Other than standard heart failure medications, there are no treatment options available targeted to maladaptive remodeling. On pathological stress, the heart reactivates a number of signaling pathways, which traditionally were thought to be operational primarily in the developing organism. The Wnt pathway is an evolutionary conserved signaling mechanism with a critical function in tumor growth 1 and cardiogenesis. 2,3 However, members of the Wnt pathway are also expressed in the normal adult heart. 4 -7 Thus, for example, the Frizzled (Fz) class of cell surface receptors of Wnt proteins, including Fz1 and Fz2, is expressed in human myocardium. 7 In infarcted hearts, Fz2 expression is considerably enhanced, 8 and in failing ventricles of humans, mRNA levels of secreted Fz-related proteins 3 and 4, which are endogenous Wnt antagonists, are elevated, leading to attenuat...