Chintan Gandhi

ADAMTS13 reduces vascular inflammation and the development of early atherosclerosis in mice

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.

ADAMTS13 deficiency exacerbates VWF-dependent acute myocardial ischemia/reperfusion injury in mice

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 VWF-mediated 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.

Alternatively-Spliced Extra Domain A of Fibronectin Promotes Acute Inflammation and Brain Injury After Cerebral Ischemia in Mice

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, an innate immune receptor that triggers proinflammatory 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 after 1-hour cerebral ischemia/23-hour reperfusion injury and compared with wild-type mice. Results— We found that EDA+/+ mice developed significantly larger infarcts and severe neurological deficits that were associated with significant increased neutrophil and macrophage infiltration as quantitated by immunohistochemistry. Additionally, we found upregulation of nuclear factor-&kgr;B, cyclo-oxygenase-2, and inflammatory cytokines tumor necrosis factor-&agr;, interleukin-1&bgr;, and interleukin-6 in the EDA+/+ mice compared with wild-type mice. Interestingly, increased brain injury and neurological deficits were largely abrogated in EDA+/+ mice by treatment with a specific Toll-like receptor 4 inhibitor. Conclusions— These findings provide the first evidence that EDA+-FN promotes inflammatory brain injury after ischemic stroke and suggest that the elevated levels of plasma EDA+-FN observed in chronic inflammatory conditions could worsen injury and outcome in patients after acute stroke.

ADAMTS13 modulates atherosclerotic plaque progression in mice via a VWF-dependent mechanism.

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 (ApoE−/−) deficient mice. It is not known whether ADAMTS13 modulates atherosclerosis directly or indirectly by cleaving ULVWF multimers.

Fibronectin Splicing Variants Containing Extra Domain A Promote Atherosclerosis in Mice Through Toll-Like Receptor 4

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 sex, 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 versus 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 NF&kgr;B-mediated inflammation (increased phospho-NF&kgr;B p65/NF&kgr;B p65, tumor necrosis factor-&agr;, and interleukin-1&bgr;) 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.