Abstract Background Soluble complement receptor‐1 (sCR1), a potent complement inhibitor, confers neuroprotection in a murine stroke model. Additional neuroprotective benefit is achieved by sLe x ‐glycosylation of sCR1. In an effort to translate sCR1‐sLe x to clinical trials, we evaluated this agent in a primate stroke model. Methods Adult male baboons randomly received either sCR1‐sLe x or vehicle. Stroke volume was assessed on day 3, and neurological examinations were conducted daily. Complement activity (CH 50 ) was measured at 30 minute, 2, 6, 12 hour, 3, and 10 days post‐ischemia. Results The experiment was terminated prematurely following an interim analysis. In a preliminary cohort (n = 3 per arm), infarct volume was greater in the treated animals. No difference in neurological score was found between groups. CH 50 levels were significantly reduced in the sCR1sLe x ‐treated groups. A hypotensive response was also observed in animals treated with sCR1‐sLe x . Conclusions Further work is necessary to explain the hypotensive response observed in primates prior to further clinical development of sCR1‐sLe x .
Endothelial CD39 metabolizes ADP released from activated platelets. Recombinant soluble human CD39 (solCD39) potently inhibited ex vivo platelet aggregation in response to ADP and reduced cerebral infarct volumes in mice following transient middle cerebral artery occlusion, even when given 3 hours after stroke. Postischemic platelet and fibrin deposition were decreased and perfusion increased without increasing intracerebral hemorrhage. In contrast, aspirin did not increase postischemic blood flow or reduce infarction volume, but did increase intracerebral hemorrhage. Mice lacking the enzymatically active extracellular portion of the CD39 molecule were generated by replacement of exons 4–6 (apyrase-conserved regions 2–4) with a PGKneo cassette. Although CD39 mRNA 3′ of the neomycin cassette insertion site was detected, brains from these mice lacked both apyrase activity and CD39 immunoreactivity. Although their baseline phenotype, hematological profiles, and bleeding times were normal, cd39–/– mice exhibited increased cerebral infarct volumes and reduced postischemic perfusion. solCD39 reconstituted these mice, restoring postischemic cerebral perfusion and rescuing them from cerebral injury. These data demonstrate that CD39 exerts a protective thromboregulatory function in stroke.
Background: Venous thrombosis (DVT) is a serious health concern, with growing incidence in an aging, comorbid population. DVT is marked by sterile inflammation and innate immune activation. Early participants in DVT include platelets, and neutrophils (PMN) which extrude their DNA to form thrombus-potentiating extracellular traps (NETs). To identify improved therapeutic targets at the nexus of inflammation and coagulation, we focused on the vascular ectonucleotidase CD39, found on leukocytes and the endothelium. CD39 dissipates extracellular ATP & ADP, thrombo-inflammatory “danger” signals. We hypothesized that CD39 is a critical enzyme in venous homeostasis, restraining unchecked inflammation and thrombosis in DVT. Methods: DVT was induced in Cd39 +/- and WT control mice using a “flow-restriction” model of inferior vena cava (IVC) stenosis. Thrombus frequency and size were assessed 48h after DVT induction. A novel flow cytometry method, immunoblot, immunostaining were used to examine thrombus cellular content, fibrin, NETs, and inflammasome activation. Results: Cd39 +/- mice had a significantly higher thrombus frequency (2.5-fold) and clot size (3-fold), with more fibrin content by immunoblot. Flow cytometry revealed exaggerated PMN recruitment to the growing thrombus in Cd39 +/- mice, with more NETs in vitro , and within DVT compared with WT mice. Cd39 +/- mice also had amplified DVT inflammasome activity in vivo , measured by increased NFkB phosphorylation and mature IL-1 beta compared with WT DVT. Flow cytometry of the thrombus revealed an increase in activated platelet-PMN heteroaggregates within Cd39 +/- DVTs, indicating enhanced innate-coagulation system crosstalk. Conclusion: CD39 is a critical vasculoprotective ecto-enzyme in venous thrombogenesis. Cd39 +/- mice have increased DVT burden, fibrin deposition, activated platelet-PMN interactions, and exaggerated inflammasome activation compared with WT mice. By enrolling NET formation and inflammasome activation, CD39 posits a previously unexplored link between venous inflammation and thrombosis. Studies are underway to modulate inflammasome activation in Cd39 +/- mice, and to delineate the relative contributions of leukocyte/endothelial CD39 in venous thrombosis.
Carbon monoxide (CO), an endogenous cytoprotective product of heme oxygenase type-1 regulates target thrombotic and inflammatory genes in ischemic stress. Regulation of the gene encoding early growth response 1 (Egr-1), a potent transcriptional activator of deleterious thrombotic and inflammatory cascades, may govern CO-mediated ischemic lung protection. The exact signaling mechanisms underlying CO-mediated cytoprotection are not well understood. In this study we tested the hypothesis that inhibition of mitogen-activated protein kinase-dependent Egr-1 expression may be pivotal in CO-mediated ischemic protection. In an in vivo isogeneic rat lung ischemic injury model, inhaled CO not only diminished fibrin accumulation and leukostasis and improved gas exchange and survival but also suppressed extracellular signal-regulated kinase (ERK) activation, Egr-1 expression, and Erg DNA-binding activity in lung tissue. Additionally, CO-mediated inhibition of Egr-1 reduced expression of target genes, such as tissue factor, serpine-1, interleukin-1, and TNF-α. However, CO failed to inhibit serpine-1 expression after unilateral lung ischemia in mice null for the Egr-1 gene. In RAW macrophages in vitro , hypoxia-induced Egr-1 mRNA expression was ERK-dependent, and CO-mediated suppression of ERK activation resulted in Egr-1 inhibition. Furthermore, CO suppression of ERK phosphorylation was reversed by the guanylate cyclase inhibitor 1 H -[1,2,4]oxadiazolo[4,3- a ]quinoxalin-1-one but was insensitive to cAMP-dependent protein kinase A inhibition with H89 and NO synthase inhibition with l -nitroarginine methyl ester. This finding indicates that CO suppresses ERK in a cGMP-dependent but cAMP/protein kinase A- and NO-independent manner. Together, these data identify a unifying molecular mechanism by which CO interrupts proinflammatory and prothrombotic mediators of ischemic injury.
Chronic airway rejection is characterized by prolonged inflammation, epithelial damage, and eventual luminal obliterative bronchiolitis (OB). In cardiac allografts, the inducible nitric oxide synthase (iNOS) promotes acute rejection but paradoxically reduces neointimal formation, the hallmark of chronic rejection. The specific roles of NOS isoforms in modulating lymphocyte traffic and airway rejection are not known. Using a double lumen mouse tracheal transplant model, tracheal grafts from B10.A (allo) or C57BL/6J (iso) mice were transplanted into cyclosporine-treated wild-type (WT) iNOS−/− or endothelial NOS (eNOS)−/− recipients. OB was observed in WT tracheal allografts at 3 weeks (53 ± 2% luminal occlusion vs. 17 ± 1% for isografts, P < 0.05) with sites of obstructive lesion formation coinciding with areas of CD3+ CD8+ T cell–rich lymphocytic bronchitis. In contrast, allografts in iNOS−/− recipients exhibited reductions in local expression of proinflammatory chemokines and cytokines, graft T cell recruitment and apoptosis, and luminal obliteration (29 ± 2%, P < 0.05 vs. WT allografts). Recipient eNOS deficiency, however, suppressed neither chemokine expression, lymphocyte infiltration, nor airway occlusion (54 ± 2%). These data demonstrate that iNOS exacerbates luminal obliteration of airway allografts in contrast with the known suppression by iNOS of cardiac allograft vasculopathy. Because iNOS−/− airways transplanted into WT allograft hosts are not protected from rejection, these data suggest that iNOS expressed by graft-infiltrating leukocytes exerts the dominant influence on airway rejection.
