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ADAMTS13, a metalloprotease, plays a pivotal role in preventing spontaneous microvascular thrombosis by cleaving hyperactive ultra large von Willebrand factor multimers into smaller, less active multimers. Reduced ADAMTS13 activity in plasma has been described in many diseases associated with systemic inflammation. It remains uncertain, however, whether ADAMTS13 contributes to disease pathogenesis or rather simply serves as an inflammation-associated marker. We hypothesized that, by decreasing vascular inflammation, ADAMTS13 reduces the development of early atherosclerotic plaques. Using intravital fluorescence microscopy, we observed excessive leukocyte adhesion and accelerated atherosclerotic plaque formation at the carotid sinus of Adamts13 ؊/؊ /ApoE ؊/؊ mice compared with ApoE ؊/؊ mice fed a high-fat Western diet. At 4 months of age, there was a significant increase in atherosclerosis in the aorta and aortic sinus of Adamts13 ؊/؊ / ApoE ؊/؊ mice compared with ApoE ؊/؊ mice. Interestingly, we detected a 2-fold increase in macrophage recruitment to the atherosclerotic plaque of the Adamts13 ؊/؊ /ApoE ؊/؊ mice compared with ApoE ؊/؊ mice, suggesting that the atherosclerotic lesions in these mice were not only larger but also more inflammatory. These findings reveal a new functional role for the antithrombotic enzyme ADAMTS13 in reducing excessive vascular inflammation and plaque formation during early atherosclerosis. (Blood. 2012; 119(10):2385-2391) Introduction ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type I repeats-13) is a zinc-containing protease that is synthesized primarily by hepatic stellate cells 1,2 and, to a lesser extent, by endothelial cells, 3 both of which secrete ADAMTS13 into the bloodstream. The only known substrate for ADAMTS13 is von Willebrand factor (VWF), a hemostatic glycoprotein that is stored as ultra large VWF (ULVWF) multimers in platelet ␣-granules and endothelial storage granules called Weibel-Palade bodies. 4 ULVWF multimers, which can be as large as 20 000 kDa and are considered to be the most hyperactive and thrombogenic form of VWF, 4,5 are not present in blood plasma under normal conditions. However, on endothelial cell activation or injury, ULVWF multimers are released into the circulation from WeibelPalade bodies. During the process of secretion, ULVWF remains transiently bound to the endothelial surface where they are cleaved by ADAMTS13 into smaller, less active VWF multimers, thereby reducing its thrombotic potential. 6 The essential role of VWF in hemostasis is evidenced clinically in patients with von Willebrand disease, a bleeding disorder caused by a lack of functional VWF. 7 In distinction, deficiency of ADAMTS13 increases plasma levels of ULVWF and causes thrombotic thrombocytopenic purpura, a disorder of thrombotic microangiopathy. 8 Several epidemiologic studies have demonstrated reduced plasma ADAMTS13 activity in inflammatory conditions, including aging, 9 systemic inflammation, 10 pancreatitis, 11 and sepsis. 12,13 It remains uncertain, ...

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...

The present study was aimed to investigate the combined effects of green tea and vitamin E on heart weight, body weight, serum marker enzymes, lipid peroxidation, endogenous antioxidants and membrane bound ATPases in isoproterenol (ISO)-induced myocardial infarction in rats. Adult male albino rats, treated with ISO (200 mg/kg, s.c.) for 2 days at an interval of 24 h caused a significant (P<0.05) elevation of heart weight, serum marker enzymes, lipid peroxidation and Ca+2 ATPase level whereas there was a significant (P<0.05) decrease in body weight, endogenous antioxidants, Na+/ K+ ATPase and Mg+2 ATPase levels. Administration of green tea (100 mg/kg/day, p.o.) and vitamin E (100 mg/kg/day, p.o.) together for 30 consecutive days and challenged with ISO on the day 29th and 30th, showed a significant (P<0.05) decrease in heart weight, serum marker enzymes, lipid peroxidation, Ca+2 ATPase and a significant increase in the body weight, endogenous antioxidants, Na+/K+ ATPase and Mg+2 ATPase when compared with ISO treated group and green tea or vitamin E alone treated groups. These findings indicate the synergistic protective effect of green tea and vitamin E during ISO induced myocardial infarction in rats.

Objective Cellular fibronectin containing extra domain A (EDA+-FN) is abundant in the arteries of patients with atherosclerosis. Several in vitro studies suggest that EDA+-FN interacts with Toll-like receptor 4 (TLR4). We tested the hypothesis that EDA+-FN exacerbates atherosclerosis through TLR4 in a clinically-relevant model of atherosclerosis, the apolipoprotein E-deficient (Apoe−/−) mouse. Approach and Results The extent of atherosclerosis was evaluated in whole aortae and cross sections of the aortic sinus in male and female EDA−/−Apoe−/− mice (which lack EDA+-FN), EDAfl/flApoe−/− mice (which constitutively express EDA+-FN) and control Apoe−/− mice fed a high-fat “Western” diet for 14 weeks. Irrespective of gender, EDAfl/flApoe−/− mice exhibited a 2-fold increase in atherosclerotic lesions (aorta and aortic sinus) and macrophage content within plaques, whereas EDA−/−Apoe−/− mice exhibited reduced atherosclerotic lesions (P<0.05 vs. Apoe−/−, n=10-12 mice/group), although cholesterol and triglyceride levels, and circulating leukocytes were similar. Genetic ablation of TLR4 partially reversed atherosclerosis exacerbation in EDAfl/flApoe−/− mice (P<0.05) but had no effect on atherosclerotic lesions in EDA−/−Apoe−/− mice. Purified cellular FN, which contains EDA, potentiated dose-dependent NFκB-mediated inflammation (increased phospho-NFκB p65/ NFκB p65, TNFα and IL1β) in bone marrow-derived macrophages from EDA−/−Apoe−/− mice but not from EDA−/−TLR4−/−Apoe−/− mice. Finally, using immunohistochemistry, we provide evidence for the first time that EDA+-FN colocalizes with macrophage TLR4 in murine aortic lesions and human coronary artery atherosclerotic plaques. Conclusions Our findings reveal that TLR4 signaling contributes to EDA+-FN mediated exacerbation of atherosclerosis. We suggest that EDA+-FN could be a therapeutic target in atherosclerosis.

Background and purpose The fibronectin isoform containing the alternatively-spliced extra domain A (EDA+-FN) is normally absent from the circulation, but plasma levels of EDA+-FN can become markedly elevated in several human pathological conditions associated with inflammation including ischemic stroke. It remains unknown whether EDA+-FN contributes to stroke pathogenesis or is simply an associative marker. Several in vitro studies suggest that EDA+-FN can activate toll-like receptor 4 (TLR4), an innate immune receptor that triggers pro-inflammatory responses. We undertook a genetic approach in mice to investigate the ability of EDA+-FN to mediate inflammatory brain damage in a focal cerebral ischemia/reperfusion injury model. Methods We used genetically modified EDA+/+ mice, which constitutively express EDA+-FN. Extent of injury, neurological outcome and inflammatory mechanisms were assessed following one hour cerebral ischemia/23 hour reperfusion injury and compared to wild-type (WT) mice. Results We found that EDA+/+ mice developed significantly larger infarcts and severe neurological deficits that was associated with significant increased neutrophil and macrophage infiltration as quantitated by immunohistochemistry. Additionally, we found upregulation of NF-κB, COX-2, and inflammatory cytokines TNFα, IL-1β and IL-6 in the EDA+/+ mice compared to WT mice. Interestingly, increased brain injury and neurological deficits were largely abrogated in EDA+/+ mice by treatment with a specific TLR4 inhibitor. Conclusions These findings provide the first evidence that EDA+-FN promotes inflammatory brain injury following ischemic stroke and suggest that the elevated levels of plasma EDA+-FN observed in chronic inflammatory conditions could worsen injury and outcome in patients following acute stroke.

Summary. Background: ADAMTS13 reduces the adhesiveness of hyperactive ultra-large von Willebrand factor (ULVWF) multimers by cleaving them into smaller, less active multimers. Recently, we and others have demonstrated that ADAMTS13 reduces atherosclerosis in hypercholesteremic apolipoprotein E (ApoEÀ/À) deficient mice. It is not known whether ADAMTS13 modulates atherosclerosis directly or indirectly by cleaving ULVWF multimers. Objective: We generated triple knockout Adamts13À/À/VwfÀ/À/ApoEÀ/À mice to determine whether ADAMTS13 modulates atherosclerosis through its proteolytic effects on VWF or other potential mechanisms. Methods: Female mice were fed a high-fat Western diet beginning at 6 weeks of age until they were sacrificed at 4 months. We compared the extent of atherosclerosis in the serial cross-sections of the aortic sinus using the Verhoeff-Van Gieson stain. Macrophage and neutrophil infiltration were quantified by immunohistochemistry. Under plain polarized light interstitial collagen content in the serial cross-sections of the aortic sinus was quantified using picrosirius red stain. Results: Deficiency of VWF in Adamts13À/À/ApoEÀ/À mice (Adamts13À/ À/VwfÀ/À/ApoEÀ/À) completely reversed exacerbated atherosclerosis (P < 0.05 vs. Adamts13À/À/ApoEÀ/À mice). The lesion size, macrophage and neutrophil infiltration in the aortic sinus of Adamts13À/À/VwfÀ/ À/ApoEÀ/À mice were significantly decreased compared with Adamts13À/À/ApoEÀ/À mice (P < 0.05), but similar to VwfÀ/À/ApoEÀ/À mice. Additionally, interstitial collagen content in the aortic sinus of Adamts13À/À/VwfÀ/ À/ApoEÀ/À mice was significantly reduced compared with Adamts13À/À/ApoEÀ/À mice (P < 0.05), but similar to VwfÀ/À/ApoEÀ/À mice. Total cholesterol and triglyceride levels were similar among groups. Conclusions: AD-AMTS13 modulates inflammatory plaque progression in hypercholesterolemic mice through a VWF-dependent mechanism. These findings provide further evidence on the pathophysiological role for the ADAMTS13/VWF axis in atherosclerosis.

Among the heart diseases, ischemia and reperfusion (I/R) induced arrhythmias contribute to episodes of sudden death. Cardiac arrhythmias during ischemia reperfusion are believed to be related to oxidative stress. Therefore, the aim of this study was to examine whether treatment with hesperidin alleviates arrhythmias and infarct size in experimentally-induced myocardial I/R injury using an in vivo rat model. In this study haemodynamics parameters, markers of inflammation, biomarkers of oxidative stress and tissue nitrite level and infarct size of the heart were estimated in various groups. I/R showed a significant decrease in tissue nitrite and antioxidant level and significant increase in arrhythmias, inflammation and myocardial cell apoptosis. Treatment with hesperidin showed a significant increase in tissue nitrite, antioxidant level and reduction in inflammation, arrhythmias and apoptosis. In conclusion, the protecting effect of hesperidin in I/R induced arrhythmias is due to reduction in inflammation and oxidative stress.