Epidemiologic studies suggest that elevated VWF levels and reduced ADAMTS13 activity in the plasma are risk factors for myocardial infarction. However, it remains unknown whether the ADAMTS13-VWF axis plays a causal role in the pathophysiology of myocardial infarction. In the present study, we tested the hypothesis that ADAMTS13 reduces VWFmediated acute myocardial ischemia/ reperfusion (I/R) injury in mice. Infarct size, neutrophil infiltration, and myocyte apoptosis in the left ventricular area were quantified after 30 minutes of ischemia and 23.5 hours of reperfusion injury. Adamts13 ؊/؊ mice exhibited significantly larger infarcts concordant with increased neutrophil infiltration and myocyte apoptosis compared with wild-type (WT) mice. In contrast, Vwf ؊/؊ mice exhibited significantly reduced infarct size, neutrophil infiltration, and myocyte apoptosis compared with WT mice, suggesting a detrimental role for VWF in myocardial I/R injury. Treating WT or Adamts13 ؊/؊ mice with neutralizing Abs to VWF significantly reduced infarct size compared with control Ig-treated mice. Finally, myocardial I/R injury in Adamts13 ؊/؊ /Vwf ؊/؊ mice was similar to that in Vwf ؊/؊ mice, suggesting that the exacerbated myocardial I/R injury observed in the setting of ADAMTS13 deficiency is VWF dependent. These findings reveal that ADAMTS13 and VWF are causally involved in myocardial I/R injury. (Blood. 2012;120(26):5224-5230) Introduction ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type I repeats-13) is a plasma protease that is synthesized primarily by hepatic stellate cells 1,2 and to a lesser extent by endothelial cells 3 and megakaryocytes. 4 The only known substrate for ADAMTS13 is VWF, a multimeric glycoprotein that plays a key role in hemostasis and thrombosis by stabilizing factor VIII and initiating platelet adhesion and aggregation at sites of vascular injury. 5 VWF is stored as ultra-large VWF (ULVWF) multimers in platelet ␣-granules and endothelial storage granules called WeibelPalade bodies. ULVWF multimers are considered hyperactive because they bind avidly to the extracellular matrix 6 and form high-strength bonds with platelet glycoprotein Ib␣ (GPIb␣). 7 ULVWF multimers are not present in the plasma of healthy humans; however, on endothelial cell activation or injury, they are released into the circulation from Weibel-Palade bodies. During the process of secretion, ULVWF multimers remain transiently bound to the endothelial surface, where they are cleaved by ADAMTS13 into smaller and less active VWF multimers. 8 Clinically, deficiency of VWF causes VWD, the most common bleeding disorder in humans. 9 Conversely, deficiency of ADAMTS13 results in accumulation of ULVWF multimers in the plasma and causes thrombotic thrombocytopenic purpura, a disorder of thrombotic microangiopathy. 10 Cardiovascular diseases, including myocardial infarction and ischemic stroke, are major causes of mortality and disability worldwide and are a major contributor to rising health care costs. Several case-control studi...

Neutrophil (polymorphonuclear leukocyte) activation with release of granule contents plays an important role in the pathogenesis of acute lung injury, prompting clinical trials of inhibitors of neutrophil elastase. Despite mounting evidence for neutrophil-mediated host tissue damage in a variety of disease processes, mechanisms regulating azurophilic granule exocytosis at the plasma membrane, and thus release of elastase and other proteases, are poorly characterized. We hypothesized that azurophilic granule exocytosis would be enhanced under priming conditions similar to those seen during acute inflammatory events and during chronic inflammatory disease, and selected the cytokine TNF-α to model this in vitro. Neutrophils stimulated with TNF-α alone elicited intracellular reactive oxygen species (ROS) generation and mobilization of secretory vesicles, specific, and gelatinase granules. p38 and ERK1/2 MAPK were involved in these components of priming. TNF-α priming alone did not mobilize azurophilic granules to the cell surface, but did markedly increase elastase release into the extracellular space in response to secondary stimulation with N-formyl-Met-Leu-Phe (fMLF). Priming of fMLF-stimulated elastase release was further augmented in the absence of NADPH oxidase-derived ROS. Our findings provide a mechanism for host tissue damage during neutrophil-mediated inflammation and suggest a novel anti-inflammatory role for the NADPH oxidase.

Deficiency of the Nox2 (gp91phox) catalytic subunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is a genetic cause of X-linked chronic granulomatous disease, a condition in which patients are prone to infection resulting from the loss of oxidant production by neutrophils. Some studies have suggested a role for superoxide derived from Nox2 NADPH oxidase in platelet activation and thrombosis, but data are conflicting. Using a rigorous and comprehensive approach, we tested the hypothesis that genetic deficiency of Nox2 attenuates platelet activation and arterial thrombosis. Our study was designed to test the genotype differences within male and female mice. Using chloromethyl-dichlorodihydrofluorescein diacetate, a fluorescent dye, as well as high-performance liquid chromatography analysis with dihydroethidium as a probe to detect intracellular reactive oxygen species (ROS), we observed no genotype differences in ROS levels in platelets. Similarly, there were no genotype-dependent differences in levels of mitochondrial ROS. In addition, we did not observe any genotype-associated differences in platelet activation, adhesion, secretion, or aggregation in male or female mice. Platelets from chronic granulomatous disease patients exhibited similar adhesion and aggregation responses as platelets from healthy subjects. Susceptibility to carotid artery thrombosis in a photochemical injury model was similar in wild-type and Nox2-deficient male or female mice. Our findings indicate that Nox2 NADPH oxidase is not an essential source of platelet ROS or a mediator of platelet activation or arterial thrombosis in large vessels, such as the carotid artery.

Hyperhomocysteinemia confers a high risk for thrombotic vascular events, but homocysteine-lowering therapies have been ineffective in reducing the incidence of secondary vascular outcomes, raising questions regarding the role of homocysteine as a mediator of cardiovascular disease. Therefore, to determine the contribution of elevated homocysteine to thrombosis susceptibility, we studied Cbs ؊/؊ mice conditionally expressing a zinc-inducible mutated human CBS (I278T) transgene. Tg-I278T Cbs ؊/؊ mice exhibited severe hyperhomocysteinemia and endothelial dysfunction in cerebral arterioles. Surprisingly, however, these mice did not display increased susceptibility to arterial or venous thrombosis as measured by photochemical injury in the carotid artery, chemical injury in the carotid artery or mesenteric arterioles, or ligation of the inferior vena cava. A survey of hemostatic and hemodynamic parameters revealed no detectible differences between control and Tg-I278T Cbs ؊/؊ mice. Our data demonstrate that severe elevation in homocysteine leads to the development of vascular endothelial dysfunction but is not sufficient to promote thrombosis. These findings may provide insights into the failure of homocysteinelowering trials in secondary prevention from thrombotic vascular events. (Blood. 2012;119(13):3176-3183) IntroductionHyperhomocysteinemia is defined as an elevation of plasma total homocysteine (tHcy) 1 and is an independent risk factor for arterial and venous vascular diseases. [2][3][4] The classic form of severe hyperhomocysteinemia arises from a rare homozygous deficiency in cystathionine ␤-synthase (CBS) which metabolizes homocysteine to cystathionine. Patients with severe hyperhomocysteinemia have plasma tHcy levels Ͼ 200M and, if untreated, ϳ 50% suffer from life-threatening thromboembolic events before the age of 30. 5 Treatment with B vitamins and betaine in conjunction with restriction of methionine improves vascular outcomes, 6 though in most patients this intervention only partially lowers tHcy levels. Therefore, the causal relationship between elevated tHcy and vascular events remains poorly defined.Mild to moderate hyperhomocysteinemia, with plasma tHcy levels of 15-50M, occurs more commonly than severe hyperhomocysteinemia and is also associated with elevated risk for myocardial infarction, stroke, and thrombosis. [2][3][4] Despite this established clinical association, treatments to lower tHcy with B vitamins paradoxically fail to decrease secondary vascular events in these patients. [7][8][9][10][11][12] This lack of benefit of homocysteine-lowering therapy is in contrast to patients with severe hyperhomocysteinemia and raises further questions regarding whether homocysteine is a mediator or biomarker of vascular disease.Both genetic and dietary animal models of hyperhomocysteinemia have been used to examine the vascular effects of elevated tHcy levels. Mouse models with genetic deficiency in CBS have been demonstrated to have endothelial dysfunction. [13][14][15] With dietary induction of hype...

Diet-induced hyperhomocysteinemia produces endothelial and cardiac dysfunction and promotes thrombosis through a mechanism proposed to involve oxidative stress. Inducible nitric oxide synthase (iNOS) is upregulated in hyperhomocysteinemia and can generate superoxide. We therefore tested the hypothesis that iNOS mediates the adverse oxidative, vascular, thrombotic, and cardiac effects of hyperhomocysteinemia. Mice deficient in iNOS (Nos2−/−) and their wild-type (Nos2+/+) littermates were fed a high methionine/low folate (HM/LF) diet to induce mild hyperhomocysteinemia, with a 2-fold increase in plasma total homocysteine (P<0.001 vs. control diet). Hyperhomocysteinemic Nos2+/+ mice exhibited endothelial dysfunction in cerebral arterioles, with impaired dilatation to acetylcholine but not nitroprusside, and enhanced susceptibility to carotid artery thrombosis, with shortened times to occlusion following photochemical injury (P<0.05 vs. control diet). Nos2−/− mice had decreased rather than increased dilatation responses to acetylcholine (P<0.05 vs. Nos2+/+ mice). Nos2−/− mice fed control diet also exhibited shortened times to thrombotic occlusion (P<0.05 vs. Nos2+/+ mice), and iNOS deficiency failed to protect from endothelial dysfunction or accelerated thrombosis in mice with hyperhomocysteinemia. Deficiency of iNOS did not alter myocardial infarct size in mice fed the control diet but significantly increased infarct size and cardiac superoxide production in mice fed the HM/LF diet (P<0.05 vs. Nos2+/+ mice). These findings suggest that endogenous iNOS protects from, rather than exacerbates, endothelial dysfunction, thrombosis, and hyperhomocysteinemia-associated myocardial ischemia-reperfusion injury. In the setting of mild hyperhomocysteinemia, iNOS functions to blunt cardiac oxidative stress rather than functioning as a source of superoxide.