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Background: Bladder cancer cells orchestrate tumour progression by pro-inflammatory cytokines. Cytokines modulate the local tumour microenvironment and increase the susceptibility of tumour distant tissues for metastasis. Here, we investigated the impact of human bladder cancer cell derived factors on the ability to modulate and activate human vascular endothelial cells., Methods: The pro-inflammatory and pro-coagulatory potential of four different bladder cancer cell lines was accessed by qRT-PCR arrays and ELISA. Modulation and activation of endothelial cells was studied in microfluidic devices. Clinical relevance of our findings was confirmed by immune histology in tissue samples of bladder cancer patients and public transcriptome data., Results: The unbalanced ratio between interleukin (IL)-1 and IL-1 receptor antagonist (IL-1ra) in the secretome of bladder cancer cells converted the quiescent vascular endothelium into a pro-adhesive, pro-inflammatory, and pro-coagulatory surface. Microfluidic experiments showed that tumour cell induced endothelial cell activation promoted leukocyte recruitment and platelet adhesion. Human bladder cancer tissue analysis confirmed that loss of IL-1ra and elevated IL-1 expression was associated with enhanced cancer progression., Conclusions: Our data indicate that IL-1 and IL-1ra were dysregulated in bladder cancer and could facilitate tumour dissemination through endothelial cell activation. Targeting the IL-1/IL-1ra axis might attenuate tumour-mediated inflammation and metastasis formation.

Cancer-related venous thromboembolisms (VTE) are associated with metastasis and reduced survival in patients with urothelial cancer of the bladder. Although previous reports suggest the contribution of tissue factor and podoplanin, the mechanistic linkage between VTE and bladder cancer cell-derived molecules is unknown. Therefore, we compared distinct procoagulant pathways in four different cell lines. In vitro findings were further confirmed by microfluidic experiments mimicking the pathophysiology of tumor blood vessels and in tissue samples of patients with bladder cancer by transcriptome analysis and immunohistology. In vitro and microfluidic experiments identified bladder cancer-derived VEGF-A as highly procoagulant because it promoted the release of von Willebrand factor (VWF) from endothelial cells and thus platelet aggregation. In tissue sections from patients with bladder cancer, we found that VWF-mediated blood vessel occlusions were associated with a poor outcome. Transcriptome data further indicate that elevated expression levels of enzymes modulating VEGF-A availability were significantly connected to a decreased survival in patients with bladder cancer. In comparison with previously postulated molecular players, we identified tumor cell-derived VEGF-A and endothelial VWF as procoagulant mediators in bladder cancer. Therapeutic strategies that prevent the VEGF-A-mediated release of VWF may reduce tumor-associated hypercoagulation and metastasis in patients with bladder cancer. IMPLICATIONS: We identified the VEGF-A-mediated release of VWF from endothelial cells to be associated with bladder cancer progression., (©2020 American Association for Cancer Research.)

Systemic lupus erythematosus (SLE), systemic sclerosis (SSc), and small vessel vasculitis are three autoimmune diseases frequently manifested in the skin. They share common pathogenic features, including production of autoantibodies, loss of tolerance to self-antigens, tissue necrosis and fibrosis, vasculopathy and activation of the coagulation system. Platelets occupy a central part within the coagulation cascade and are well-recognized for their hemostatic role. However, recent cumulative evidence implicates their additional and multifaceted immunoregulatory functions. Platelets express immune receptors and they store growth factors, cytokines, and chemokines in their granules enabling a significant contribution to inflammation. A plethora of activating triggers such as damage associated molecular patterns (DAMPs) released from damaged endothelial cells, immune complexes, or complement effector molecules can mediate platelet activation. Activated platelets further foster an inflammatory environment and the crosstalk with the endothelium and leukocytes by the release of immunoactive molecules and microparticles. Further insight into the pathogenic implications of platelet activation will pave the way for new therapeutic strategies targeting autoimmune diseases. In this review, we discuss the inflammatory functions of platelets and their mechanistic contribution to the pathophysiology of SSc, ANCA associated small vessel vasculitis and other autoimmune diseases affecting the skin.

Increased levels of the chemokine CCL2 in cancer patients are associated with poor prognosis. Experimental evidence suggests that CCL2 correlates with inflammatory monocyte recruitment and induction of vascular activation, but the functionality remains open. Here, we show that endothelial Ccr2 facilitates pulmonary metastasis using an endothelial-specific Ccr2-deficient mouse model (Ccr2 ec KO). Similar levels of circulating monocytes and equal leukocyte recruitment to metastatic lesions of Ccr2 ec KO and Ccr2 fl/fl littermates were observed. The absence of endothelial Ccr2 strongly reduced pulmonary metastasis, while the primary tumor growth was unaffected. Despite a comparable cytokine milieu in Ccr2 ec KO and Ccr2 fl/fl littermates the absence of vascular permeability induction was observed only in Ccr2 ec KO mice. CCL2 stimulation of pulmonary endothelial cells resulted in increased phosphorylation of MLC2, endothelial cell retraction, and vascular leakiness that was blocked by an addition of a CCR2 inhibitor. These data demonstrate that endothelial CCR2 expression is required for tumor cell extravasation and pulmonary metastasis. IMPLICATIONS: The findings provide mechanistic insight into how CCL2-CCR2 signaling in endothelial cells promotes their activation through myosin light chain phosphorylation, resulting in endothelial retraction and enhanced tumor cell migration and metastasis., (©2018 American Association for Cancer Research.)

The dynamic change from a globular conformation to an elongated fiber determines the ability of von Willebrand factor (VWF) to trap platelets. Fiber formation is favored by the anchorage of VWF to the endothelial cell surface, and VWF-platelet aggregates on the endothelium contribute to inflammation, infection, and tumor progression. Although P-selectin and ανβ3-integrins may bind VWF, their precise role is unclear, and additional binding partners have been proposed. In the present study, we evaluated whether the endothelial glycocalyx anchors VWF fibers to the endothelium. Using microfluidic experiments, we showed that stabilization of the endothelial glycocalyx by chitosan oligosaccharides or overexpression of syndecan-1 (SDC-1) significantly supports the binding of VWF fibers to endothelial cells. Heparinase-mediated degradation or impaired synthesis of heparan sulfate (HS), a major component of the endothelial glycocalyx, reduces VWF fiber-dependent platelet recruitment. Molecular interaction studies using flow cytometry and live-cell fluorescence microscopy provided further evidence that VWF binds to HS linked to SDC-1. In a murine melanoma model, we found that protection of the endothelial glycocalyx through the silencing of heparanase increases the number of VWF fibers attached to the wall of tumor blood vessels. In conclusion, we identified HS chains as a relevant binding factor for VWF fibers at the endothelial cell surface in vitro and in vivo., (© 2018 by The American Society of Hematology.)

Co-stimulation via CD154 binding to CD40, pivotal for both innate and adaptive immunity, may also link haemostasis to vascular remodelling. Here we demonstrate that human platelet-bound or recombinant soluble CD154 (sCD154) elicit the release from and tethering of ultra-large (UL) von Willebrand factor (vWF) multimers to the surface of human cultured endothelial cells (ECs) exposed to shear stress. This CD40-mediated ULVWF multimer release from the Weibel-Palade bodies was triggered by consecutive activation of TRAF6, the tyrosine kinase c-Src and phospholipase Cγ1 followed by inositol-1,4,5 trisphosphate-mediated calcium mobilisation. Subsequent exposure to human washed platelets caused ULVWF multimer-platelet string formation on the EC surface in a shear stress-dependent manner. Platelets tethered to these ULVWF multimers exhibited P-selectin on their surface and captured labelled monocytes from the superfusate. When exposed to shear stress and sCD154, native ECs from wild-type but not CD40 or vWF-deficient mice revealed a comparable release of ULVWF multimers to which murine washed platelets rapidly adhered, turning P-selectin-positive and subsequently capturing monocytes from the perfusate. This novel CD154-provoked ULVWF multimer-platelet string formation at normal to fast flow may contribute to vascular remodelling processes requiring the perivascular or intravascular accumulation of pro-inflammatory macrophages such as arteriogenesis or atherosclerosis.

Background: During pathogenesis of infective endocarditis, Staphylococcus aureus adherence often occurs without identifiable preexisting heart disease. However, molecular mechanisms mediating initial bacterial adhesion to morphologically intact endocardium are largely unknown., Methods and Results: Perfusion of activated human endothelial cells with fluorescent bacteria under high-shear-rate conditions revealed 95% attachment of the S aureus by ultralarge von Willebrand factor (ULVWF). Flow experiments with VWF deletion mutants and heparin indicate a contribution of the A-type domains of VWF to bacterial binding. In this context, analyses of different bacterial deletion mutants suggest the involvement of wall teichoic acid but not of staphylococcal protein A. The presence of inactivated platelets and serum increased significantly ULVWF-mediated bacterial adherence. ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin motifs 13) caused a dose-dependent reduction of bacterial binding and a reduced length of ULVWF, but single cocci were still tethered by ULVWF at physiological levels of ADAMTS13. To further prove the role of VWF in vivo, we compared wild-type mice with VWF knockout mice. Binding of fluorescent bacteria was followed in tumor necrosis factor-α-stimulated tissue by intravital microscopy applying the dorsal skinfold chamber model. Compared with wild-type mice (n=6), we found less bacteria in postcapillary (60±6 versus 32±5 bacteria) and collecting venules (48±5 versus 18±4 bacteria; P<0.05) of VWF knockout mice (n=5)., Conclusions: Our data provide the first evidence that ULVWF contributes to the initial pathogenic step of S aureus-induced endocarditis in patients with an apparently intact endothelium. An intervention reducing the ULVWF formation with heparin or ADAMTS13 suggests novel therapeutic options to prevent infective endocarditis.

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.

Reperfusion after ischemic conditions induces massive endothelial cell (EC) activation, an initial step of reperfusion injury. Reperfusion is characterized by reoxygenation, realkalinization and a localized increase of inflammatory stimuli. In this study, we focused on the influence of extracellular realkalinization on human umbilical vein endothelial cell (HUVEC) activation. We examined intracellular pH (pH(in)) and intracellular free calcium concentration ([Ca(2+)](in)), a second messenger known to mediate von Willebrand factor (VWF) exocytosis in endothelium, upon realkalinization. Furthermore, we measured the agonist-stimulated exocytosis of VWF, Interleukin-8 and soluble P-selectin (sP-Selectin) as markers of EC activation. To verify a morphological correlate of EC activation, we finally observed platelet-endothelial adherence during realkalinization using shear flow. Realkalinization of HUVEC was simulated by switching from bicarbonate buffered Ringer solution of an acidotic pH(ex) of 6.4 to a physiologic pH(ex) of 7.4. Extracellular realkalinization was accompanied by pH(in) recovery from 6.5 to 7.2 within 10 min. Application of cariporide, an inhibitor of the Na(+)/H(+) exchanger subtype 1 (NHE), during extracellular realkalinization significantly delayed the early kinetics of intracellular realkalinization. Histamine stimulated [Ca(2+)](in) was significantly increased upon realkalinization compared to control cells. Also agonist-stimulated release of VWF, Interleukin-8 and sP-Selectin was massively enhanced during pH(in) recovery in comparison to control. Furthermore, we observed an increased platelet binding to endothelium. Interestingly, each of these realkalinization-induced effects were significantly reduced by early application of cariporide. Therefore, delay of acute NHE-dependent pH(in) recovery may represent a promising mechanism for inhibition of EC activation upon reperfusion., ((c) 2007 Wiley-Liss, Inc.)

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.

Atopic dermatitis (AD) lesions are characterized by differences in the activation state of endothelial cells and vascular smooth muscle cells and the release of inflammatory mediators by and toward the vasculature. The vascular system, including endothelial cells and smooth muscle cells, is ultimately involved in clinical symptoms of AD, such as erythema, edema, leukocyte recruitment, and white dermographism. Various mediators and bidirectional neurovascular interactions regulate the inflammatory response during AD. T cell-endothelial cell interactions are a crucial component to establish acute AD. Various immune cells, including monocytes and mast cells, communicate with the endothelium by releasing inflammatory mediators, thereby stimulating inflammatory mediator release from activated endothelial cells. The process of adhesion, tethering, and transmigration of infiltrating cells is a highly regulated and an active communication process between endothelial cells and leukocytes. Endothelial cells play a pivotal role in the pathophysiology of AD and represent future targets for the treatment of AD.

Objective: 17beta-estradiol is known to delay the onset of atherosclerosis in women but cellular mechanisms are still unclear. Estrogens bind to specific receptors and initiate a signaling cascade that involves the activation of plasma membrane Na(+)/H(+) exchange. We hypothesized that estrogens interfere with ion transport across the plasma membrane and thus control endothelial structure and function. Therefore, we investigated the effects of the sex steroids 17beta-estradiol, progesterone, and testosterone on volume, apical surface and elasticity in human endothelium., Methods: The atomic force microscope was used as an imaging tool and as an elasticity sensor. We applied the antiestrogen tamoxifen, the Na(+)/H(+) exchange blocker cariporide and the epithelial Na(+)channel blocker amiloride to elucidate the role of transmembrane ion transport in hormone-treated human umbilical vein endothelial cells (HUVEC)., Results: Incubation with 17beta-estradiol for 72 h led to a dose-dependent increase of endothelial cell volume (41%), apical cell surface (22%), and cell elasticity (53%) as compared to non-17beta-estradiol treated controls. Block of the 17beta-estradiol receptor by tamoxifen and of plasma membrane Na(+)/H(+) exchange by cariporide prevented the hormone-induced changes. Progesterone and testosterone were ineffective., Conclusions: 17beta-estradiol increases HUVEC water content and HUVEC elasticity mediated by activated estrogen receptors. The estrogen response depends on the activation of plasma membrane Na(+)/H(+) exchange. The increase in endothelial cell elasticity could be one of the vasoprotective mechanisms postulated for 17beta-estradiol.

There is accumulating evidence that mineralocorticoids not only act on kidney but also on the cardiovascular system. We investigated the response of human umbilical venous endothelial cells (HUVECs) to aldosterone at a time scale of 20 minutes in absence and presence of the aldosterone antagonist spironolactone or other transport inhibitors. We applied atomic force microscopy (AFM), which measures cell volume and volume shifts between cytosol and cell nucleus. We observed an immediate cell volume increase (about 10%) approximately 1 min after addition of aldosterone (0.1 micromol/l), approaching a maximum (about 18%) 10 min after aldosterone treatment. Cell volume returned to normal 20 min after hormone exposure. Spironolactone (1 micromol/l) or amiloride (1 micromol/l) prevented the late aldosterone-induced volume changes but not the immediate change observed 1 min after hormone exposure. AFM revealed nuclear swelling 5 min after aldosterone addition, followed by nuclear shrinkage 15 min later. The Na(+)/H(+) exchange blocker cariporide (10 micromol/l) was ineffective. We conclude: (i). Aldosterone induces immediate (1 min) swelling independently of plasma membrane Na(+) channels and intracellular mineralocorticoid receptors followed by late mineralocorticoid receptor- and Na(+)-channel-dependent swelling. (ii). Intracellular macromolecule shifts cause the changes in cell volume. (iii). Both amiloride and spironolactone may be useful for medical applications to prevent aldosterone-induced vasculopathies.