Background The fibronectin splicing variant containing extra domain A (Fn-EDA) is present in negligible amounts in the plasma of healthy humans, but markedly elevated in patients with comorbid conditions including diabetes and hypercholesterolemia, which are risk factors for stroke. It remains unknown, however, whether Fn-EDA worsens stroke outcomes in such conditions. We determined the role of Fn-EDA in stroke outcome in a model of hypercholesterolemia, the apolipoprotein E-deficient (Apoe−/−) mouse. Methods and Results In a transient cerebral ischemia/reperfusion injury model, Apoe−/− mice expressing Fn deficient in EDA (Fn-EDA−/−Apoe−/− mice) exhibited smaller infarcts and improved neurological outcomes at days 1 and 8 (P<0.05 vs. Apoe−/− mice). Concomitantly, intracerebral thrombosis (assessed by fibrin (ogen) deposition) and postischemic inflammation (phospho-NF-κB p65, phospho IKKα/β, IL1-β and TNFα) within lesions of Fn-EDA−/−Apoe−/− mice were markedly decreased (P<0.05 vs. Apoe−/− mice). In a FeCl3 injury-induced carotid artery thrombosis model, thrombus growth rate and the time to occlusion were prolonged in Fn-EDA−/−Apoe−/− mice (P<0.05 vs. Apoe−/− mice). Genetic ablation of TLR4 improved stroke outcome in Apoe−/− mice (P<0.05) but had no effect on stroke outcome in Fn-EDA−/−Apoe−/− mice. Bone marrow transplantation experiments revealed that non-hematopoietic cell-derived Fn-EDA exacerbates stroke through TLR4 expressed on hematopoietic cells. Infusion of a specific inhibitor of Fn-EDA into Apoe−/− mouse 15 minutes after reperfusion significantly improved stroke outcome. Conclusions Hypercholesterolemic mice deficient in Fn-EDA exhibit reduced cerebral thrombosis and less inflammatory response after ischemia/reperfusion injury. These findings suggest that targeting Fn-EDA could be an effective therapeutic strategy in stroke associated with hypercholesterolemia.
Triacylglycerol (TG) accumulation caused by adipose triglyceride lipase (ATGL) deficiency or very low-density lipoprotein (VLDL) loading of wild-type (Wt) macrophages results in mitochondrial-mediated apoptosis. This phenotype is correlated to depletion of Ca2+ from the endoplasmic reticulum (ER), an event known to induce the unfolded protein response (UPR). Here, we show that ER stress in TG-rich macrophages activates the UPR, resulting in increased abundance of the chaperone GRP78/BiP, the induction of pancreatic ER kinase-like ER kinase, phosphorylation and activation of eukaryotic translation initiation factor 2A, the translocation of activating transcription factor (ATF)4 and ATF6 to the nucleus and the induction of the cell death executor CCAAT/enhancer-binding protein homologous protein. C16:0 ceramide concentrations were increased in Atgl–/– and VLDL-loaded Wt macrophages. Overexpression of ceramide synthases was sufficient to induce mitochondrial apoptosis in Wt macrophages. In accordance, inhibition of ceramide synthases in Atgl–/– macrophages by fumonisin B1 (FB1) resulted in specific inhibition of C16:0 ceramide, whereas intracellular TG concentrations remained high. Although the UPR was still activated in Atgl–/– macrophages, FB1 treatment rescued Atgl–/– macrophages from mitochondrial dysfunction and programmed cell death. We conclude that C16:0 ceramide elicits apoptosis in Atgl–/– macrophages by activation of the mitochondrial apoptosis pathway.
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.
Objective Von Willebrand factor (VWF), which is synthesized in endothelial cells and megakaryocytes, is known to worsen stroke outcome. In vitro studies suggest that platelet-derived VWF is biochemically different from the endothelial cell-derived VWF. However, little is known about relative contribution of different pools of VWF in stroke. Approach and Results Using bone marrow transplantation, we generated chimeric platelet derived-VWF mice (Plt-VWF), platelet derived-VWF mice that lack ADAMTS13 in platelets and plasma (Plt-VWF/Adamts13−/−), and endothelial cell derived-VWF mice (EC-VWF) to determine relative contribution of different pools of VWF in stroke. In brain ischemia/reperfusion injury model, we found that infarct size, post-ischemic intracerebral thrombo-inflammation (fibrin(ogen) deposition, neutrophil infiltration, IL-1β and TNF-α levels) within lesions were comparable between EC-VWF and WT mice. Infarct size and post-ischemic thrombo-inflammation were comparable between Plt-VWF and Plt-VWF/Adamts13−/− mice, but decreased compared to EC-VWF and/or WT mice (P<0.05) and increased compared to Vwf −/− mice (P<0.05). Susceptibility to FeCl3 injury-induced carotid artery thrombosis was comparable between WT and EC-VWF mice, whereas Plt-VWF and Plt-VWF/Adamts13−/− mice exhibited defective thrombosis. Although most of the injured vessels did not occlude, slope over time showed that thrombus growth rate was increased in both Plt-VWF and Plt-VWF/Adamts13−/− mice compared to Vwf −/− mice (P<0.05), but decreased compared to WT or EC-VWF mice. Conclusions Platelet-derived VWF, either in presence or absence of ADAMTS13, partially contributes to VWF-dependent injury and post-ischemic thrombo-inflammation following stroke. Endothelial cell-derived VWF is the major determinant that mediates VWF-dependent ischemic stroke by promoting post-ischemic thrombo-inflammation.
Resting platelets rely on oxidative phosphorylation (OXPHOS) and aerobic glycolysis (conversion of glucose to lactate in the presence of oxygen) to generate adenosine triphosphate, whereas activated platelets exhibit a high level of aerobic glycolysis, suggesting the existence of metabolic flexibility in platelets. Mitochondrial pyruvate dehydrogenase kinases (PDK 1-4) play a pivotal role in metabolic flexibility by inhibiting pyruvate dehydrogenase complex. We determined whether metabolic reprogramming, diverting metabolism from aerobic glycolysis back to OXPHOS, would inhibit platelet function. PDKs activity in human and mouse platelets was inhibited with dichloroacetic acid (DCA), a potent inhibitor of all 4 forms of PDK. Human and mouse platelets pretreated with DCA exhibited decreased platelet aggregation to suboptimal doses of collagen, convulxin, thrombin, and adenosine diphosphate concomitant with decreased glucose uptake. Bioenergetics profile revealed that platelets pretreated with DCA exhibited decreased aerobic glycolysis in response to convulxin only. Furthermore, DCA inhibited ATP secretion, thromboxane A2 generation, and tyrosine phosphorylation of Syk and PLCγ2 in response to collagen or convulxin in human and mouse platelets ( < .05 vs vehicle treated). In the flow chamber assay, human and mouse blood pretreated with DCA formed smaller thrombi when perfused over collagen for 10 minutes at an arterial shear rate of 1500 s ( < .05 vs control). Wild-type mice pretreated with DCA were less susceptible to thrombosis in the FeCl-induced carotid and laser injury-induced mesenteric artery thrombosis models ( < .05 vs vehicle control), without altering hemostasis. Targeting metabolic plasticity with DCA may be explored as a novel strategy to inhibit platelet function.
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