Abstract-Three 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (HCRIs), atorvastatin, pravastatin, and cerivastatin, inhibited phorbol ester-stimulated superoxide anion (O 2 Ϫ ) formation in endothelium-intact segments of the rat aorta in a time-and concentration-dependent manner (maximum inhibition of 70% after 18 hours at 1 to 10 mol/L). The HMG-CoA reductase product mevalonic acid (400 mol/L) reversed the inhibitory effect of the HCRIs, which, conversely, was mimicked by inactivation of p21 Rac with Clostridium sordellii lethal toxin but not by inactivation of p21 Rho with Clostridium botulinum exoenzyme (C3). A mevalonate-sensitive inhibition of phorbol ester-stimulated O 2 Ϫ formation by atorvastatin was also observed in porcine cultured endothelial cells and in a murine macrophage cell line. In the rat aorta, no effect of the HCRIs on protein kinase C, NADPH oxidase, or superoxide dismutase (SOD) activity and expression was detected, whereas that of endothelial nitric oxide (NO) synthase was enhanced Ϸ2-fold. Moreover, exposure of the segments to atorvastatin resulted in a significant improvement of endothelium-dependent NO-mediated relaxation, and this effect was abolished in the presence of SOD. Taken together, these findings suggest that in addition to augmenting endothelial NO synthesis, HCRIs inhibit endothelial O 2 Ϫ formation by preventing the isoprenylation of p21 Rac, which is critical for the assembly of NADPH oxidase after activation of protein kinase C. The resulting shift in the balance between NO and O 2 Ϫ in the endothelium improves endothelial function even in healthy blood vessels and therefore may provide a reasonable explanation for the beneficial effects of HCRIs in patients with coronary heart disease in addition to or as an alternative to the reduction in serum LDL cholesterol. Key Words: HMG-CoA reductase inhibitor(s) Ⅲ endothelial dysfunction Ⅲ coronary heart disease Ⅲ nitric oxide Ⅲ superoxide anion Ⅲ NADPH oxidase Ⅲ p21 Rac T he beneficial effects of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (HCRIs), collectively referred to as "statins," on coronary events have generally been attributed to the well-documented LDL cholesterol-lowering properties of these drugs. However, despite the strong association between drug concentration, blood lipid level, and patient benefit shown in the CARE, 4S, and WOSCOPS trials, HCRIs seem to exert at least part of their cardioprotective action by mechanisms other than simply lowering the lipid load of the vessel wall. Thus, HCRIs induce regression of atherosclerotic lesions in patients with coronary heart disease (CHD) with and without hypercholesterolemia 1 and improve endothelial dysfunction, a hallmark of atherosclerotic blood vessels, in patients with moderately elevated total serum cholesterol within 1 month. 2 At the cellular level, they modulate leukocyte activity and adhesiveness, 3,4 inhibit vascular smooth muscle cell proliferation both in vitro and in vivo, 5,6 and reduce the synthesis of che...

Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung, Basel Cardiovascular Research Foundation, and B Braun Medical AG.

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...

We investigated the hypothesis that the antiatherosclerotic effect of 17beta-estradiol (E2) is due to a shift in the nitric oxide (NO)/superoxide (O2-) balance in the vessel wall, thereby increasing the bioavailability of NO. In human umbilical vein cultured endothelial cells, E2 (1-100 nmol/l), but not 17alpha-estradiol, caused a time- and concentration-dependent decrease in expression of the NADPH oxidase subunit gp91phox (up to 60% inhibition at both the mRNA and protein level). This effect was prevented by coincubation with the estrogen receptor antagonists tamoxifen and ICI 182,780 (1 micromol/l each). Within the same concentration range, E2 also up-regulated endothelial nitric oxide synthase expression ( approximately twofold). Moreover, preincubation of the cells with E2 or a gp91phox antisense oligonucleotide significantly decreased their capacity to generate O2- on phorbol ester stimulation (i.e., assembly of the active NADPH oxidase complex). Blockade of NO synthase activity, on the other hand, had no effect on phorbol ester-stimulated O2- formation. In addition, E2 (100 nmol/l) inhibited the increase in adhesion molecule and chemokine expression in cells exposed to cyclic strain. Cyclic strain enhanced endothelial O2- formation, thereby offsetting the inhibitory effect of NO on the expression of these gene products. E2 thus seems to act as an antioxidant at the genomic level which by improving the NO/O2- balance normalizes expression of proatherosclerotic gene products in endothelial cells.