Vascular injury has emerged as a complication contributing to morbidity in coronavirus disease 2019 (COVID-19). The glycosaminoglycan hyaluronan (HA) is a major component of the glycocalyx, a protective layer of glycoconjugates that lines the vascular lumen and regulates key endothelial cell functions. During critical illness, as in the case of sepsis, enzymes degrade the glycocalyx, releasing fragments with pathologic activities into circulation and thereby exacerbating disease. Here, we analyzed levels of circulating glycosaminoglycans in 46 patients with COVID-19 ranging from moderate to severe clinical severity and measured activities of corresponding degradative enzymes. This report provides evidence that the glycocalyx becomes significantly damaged in patients with COVID-19 and corresponds with severity of disease. Circulating HA fragments and hyaluronidase, 2 signatures of glycocalyx injury, strongly associate with sequential organ failure assessment scores and with increased inflammatory cytokine levels in patients with COVID-19. Pulmonary microvascular endothelial cells exposed to COVID-19 milieu show dysregulated HA biosynthesis and degradation, leading to production of pathological HA fragments that are released into circulation. Finally, we show that HA fragments present at high levels in COVID-19 patient plasma can directly induce endothelial barrier dysfunction in a ROCK- and CD44-dependent manner, indicating a role for HA in the vascular pathology of COVID-19.
Introduction: IFITM3, an interferon (IFN) responsive gene, restricts pathogen replication through vesicular trafficking mechanisms. IFITM3 in megakaryocytes (MKs) and platelets has not been examined. We hypothesized that in MKs and platelets, IFITM3 regulates the endocytosis of pro-coagulant proteins, aggregation, and thrombosis. Methods: the regulation of IFITM3 gene expression was determined in MKs and platelets under inflammatory stimuli. Fibrinogen (Fgn) endocytosis, a clathrin-mediated event requiring αIIbβ3, and transferrin endocytosis, a clathrin-mediated, αIIbβ3-independent event, were examined in stimulated MKs and platelets from wild type (WT) and Ifitm -/- mice (KO). Integrin αIIbβ3 activation, platelet aggregation, and thrombosis was determined in WT and Ifitm -/- mice upon IFNα-stimulation. Co-immunoprecipitation identified interaction partners for IFITM3. To establish human relevance, IFITM3 expression, Fgn content, and aggregation was measured in platelets from septic patients, where systemic IFNs are increased. Results: IFNs significantly induced IFITM3 expression in MKs and platelets (p<0.001). Upregulation of IFITM3 by IFNs increased Fgn and transferrin endocytosis in MKs (2-fold vs. NT, p<0.05). Co-IP demonstrated a specific IFITM3 interaction between clathrin and αIIb. Upon IFN stimulation, IFITM3, clathrin, and αIIb shifted into lipid rafts. Increased Fgn endocytosis by IFNs enhanced platelet aggregation and accelerated death (~2-fold increased) due to platelet-dependent thrombosis (p<0.05 for all comparisons). In KO mice, enhanced Fgn endocytosis, platelet aggregation, and thrombosis to IFNs was completely prevented, demonstrating the necessity of IFITMs for these functional responses. Platelets from septic patients mirrored findings in mice with a ~20-fold increase in IFITM3 protein expression, with shifts into lipid rafts, compared to healthy controls (p<0.0001). Increased platelet IFITM3 expression was associated with greater platelet Fgn content and significant increases in platelet aggregation (20% increase, p<0.05). Conclusions: Our data identify IFITM3 as a previously unknown regulator of MK and platelet endocytosis and thrombosis under inflammatory stimuli in humans and mice.
Abstract: The analgesic effect of preoperatively administered ibuprofen was evaluated in 107 dental outpatients undergoing the removal of impacted third molars. Subjects were given 800 mg ibuprofen prior to the procedure and 400 mg ibuprofen 4 and 8 hours later. Comparison was made to groups receiving either placebo at all three doses, 600 mg acetaminophen administered on the same schedule, or preoperatively administered placebo followed by two doses of postoperatively administered 600 mg acetaminophen plus 60 mg codeine. Ibuprofen pretreatment resulted in significantly less pain than placebo or acetaminophen pretreatment as the local anesthetic wore off. Ibuprofen also resulted in less postoperative pain than acetaminophen plus codeine following the second dose. Side effects were similar across drug teatments and placebo with the exception of greater reports of drowsiness following the opiate—analgesic combination. These findings indicate that pretreatment with a nonsteroidal antiinflammatory drug, such as ibuprofen, results in a suppression of postoperative pain when compared to standard therapy without an increase in side effects.
Rationale: Ischemic stroke prompts an inflammatory response which is associated with worse outcomes. Classic anti-inflammatory strategies were unsuccessful in clinical trials, implying other mechanisms contribute to injurious inflammation in stroke. In response to stimuli, neutrophils can release DNA web-like structures called neutrophil extracellular traps (NETs). Recently, a role for NETs in cardiovascular disease has emerged. Here, we studied whether NETs contribute to ischemic stroke outcomes. Methods: Markers of NET formation were assessed in brain tissue and plasma from ischemic stroke patients. For murine studies, we used male and female mice that were subjected to transient middle cerebral artery occlusion. Stroke outcomes were assessed 24 hours or 7 days after stroke. Results: NETs were found in brain tissue from deceased ischemic stroke patients. Ischemic stroke patients had significantly increased plasma biomarkers of NET formation including citrullinated histone H3 (p<0.0001) and MPO-DNA complexes (p<0.001) compared to matched controls. NET biomarkers positively correlated with worse stroke outcomes at discharge (p<0.05) while they did not correlate with stroke severity at admission. To target NET formation in ischemic stroke, we investigated the therapeutic potential of a recently discovered neonatal NET inhibitory factor (nNIF). nNIF specifically blocks NET formation in human and murine neutrophils without affecting other critical neutrophil functions. Mice prophylactically treated with nNIF had significantly reduced brain and plasma NETs after stroke while cerebral neutrophil recruitment remained unaffected. The reduction in NET formation was associated with significantly reduced neuronal apoptosis and smaller brain infarcts (p<0.0001). Furthermore, nNIF treated mice had improved neurological behavior and motor function, and enhanced 7-day survival after ischemic stroke (p<0.001). Importantly, these results were confirmed in diabetic mice and >18-month-old mice and nNIF was still effective when administered therapeutically, 1 hour after stroke onset. Conclusions: Our results support a pathological role for NETs in ischemic stroke and warrant further investigation into nNIF to improve stroke outcomes.
Through the ACE2, a main enzyme of the renin-angiotensin system (RAS), SARS-CoV-2 gains access into the cell, resulting in different complications which may extend beyond the RAS and impact the Arachidonic Acid (ArA) pathway. The contribution of the RAS through ArA pathways metabolites in the pathogenesis of COVID-19 is unknown. We investigated whether RAS components and ArA metabolites can be considered biomarkers of COVID-19. We measured the plasma levels of RAS and ArA metabolites using an LC-MS/MS. Results indicate that Ang 1-7 levels were significantly lower, whereas Ang II levels were higher in the COVID-19 patients than in healthy control individuals. The ratio of Ang 1-7/Ang II as an indicator of the RAS classical and protective arms balance was dramatically lower in COVID-19 patients. There was no significant increase in inflammatory 19-HETE and 20-HETE levels. The concentration of EETs was significantly increased in COVID-19 patients, whereas the DHETs concentration was repressed. Their plasma levels were correlated with Ang II concentration in COVID-19 patients. In conclusion, evaluating the RAS and ArA pathway biomarkers could provide helpful information for the early detection of high-risk groups, avoid delayed medical attention, facilitate resource allocation, and improve patient clinical outcomes to prevent long COVID incidence.
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