Key Points Heme, released from hemoglobin, elicits vaso-occlusion in transgenic sickle mice via endothelial TLR4 signaling. Heme/TLR4 signaling activates NF-κB and triggers vaso-occlusion through Weibel-Palade body degranulation and adhesion molecule expression.

Abnormal tissue factor (TF) expression has been demonstrated on blood monocytes and circulating endothelial cells in humans with sickle cell anemia. We have now studied sickle transgenic mice to help define the biology of endothelial TF expression in sickle disease. Using immunostaining of tissue sections, we find that this is confined almost exclusively to the pulmonary veins. About 15% and 13% of these exhibit TF-positive endothelium in the wild-type normal mouse and the normal human hemoglobin (HbA)-expressing control transgenic mouse, respectively. The mild sickle mouse is indistinguishable from normal (ϳ 14% positive), but TF expression is significantly elevated in the moderate and severe mouse models of sickle disease (ϳ 29% and ϳ 41% positive, respectively). Exposure of the mild sickle mouse to hypoxia for 3 hours, followed by reoxygenation, converted its TF ex

Genetic differences in endothelial biology could underlie development of phenotypic heterogeneity among persons afflicted with vascular diseases. We obtained blood outgrowth endothelial cells from 20 subjects with sickle cell anemia (age, 4-19 years) shown to be either at-risk (n ‫؍‬ 11) or not-at-risk (n ‫؍‬ 9) for ischemic stroke because of, respectively, having or not having occlusive disease at the circle of Willis. Gene expression profiling identified no significant single gene differences between the 2 groups, as expected. However, analysis of Biological Systems Scores, using gene sets that were predetermined to survey each of 9 biologic systems, showed that only changes in inflammation signaling are characteristic of the at-risk subjects, as supported by multiple statistical approaches. Correspondingly, subsequent biologic testing showed significantly exaggerated RelA activation on the part of blood outgrowth endothelial cells from the at-risk subjects in response to stimulation with interleukin-1␤/tumor necrosis factor␣. We conclude that the pathobiology of circle of Willis disease in the child with sickle cell anemia predominantly involves inflammation biology, which could reflect differences in genetically determined endothelial biology that account for differing host responses to inflammation. IntroductionMany human diseases present in a clinically variable manner, yet the basis for the biologic phenomenon of phenotypic heterogeneity, the variation in presentation of any given disease, is generally unknown. We have used a specific example of this phenomenon to address our overarching hypothesis that genetic, inherited differences in endothelial biology can underlie the phenotypic heterogeneity of human vascular disease.Sickle cell anemia, caused by inherited homozygosity for the mutant sickle hemoglobin, is a disease characterized by anemia, vascular occlusions, and chronic organ damage. It has an exceedingly complex pathophysiology and incredibly diverse clinical complications. 1 Among these, there are 3 stroke syndromes: clinically silent strokes occurring in children resulting from multifocal small vessel disease; hemorrhagic strokes occurring in adults; and clinical ischemic stroke, the classical stroke syndrome of sickle cell anemia.Notably, approximately 10% of children with sickle cell anemia develop classic ischemic stroke, with peak age being approximately 5 years. 2,3 Risk factors include elevated white count, low blood hemoglobin, hypertension, and a prior neurologic event. [2][3][4][5] However, the primary risk factor is occlusive disease at the circle of Willis (CoW), 6,7 the encircling structure of medium to large vessels at the base of the brain. CoW disease is thought to be causal, as the strokes tend to be due to thrombosis occurring over the area of vessel wall abnormality, and the extent of stroke correlates with degree of CoW stenosis. 2,8 Stroke pathogenesis does not simply involve sickling in the vasa vasorum because vessels in the CoW do not have vasa vasorum. 9 Our hypothesis ...

Sickle cell anemia is accompanied by activation of coagulation and thrombosis. We have studied the abnormal expression of tissue factor (TF) by the pulmonary vein endothelium of the mild-phenotype NY1DD sickle transgenic. As detected by immunofluorescence microscopy, this appears only after the NY1DD mouse is exposed to hypoxia/reoxygenation (H/R), which actually causes ischemia/ reperfusion in the sickle-but not the normal-mouse. We tested the hypothesis that the NFκB-activating inflammation that develops in post-H/R NY1DD mice is responsible for this phenotype switch. Various NFκB inhibitors (including p50-specific andrographolide) demonstrated that endothelial TF positivity is NFκB dependent. Several systemic inflammatory stimulators (TNFα, lipopolysaccharide, thioglycollate, carageenan) given to control mice showed that inflammatory promotion of TF expression by only pulmonary vein endothelium is not specific to the sickle model. We bred the NFκB(p50)−/− state into the NY1DD mouse. Combined with marrow transplantation, this allowed creation of NY1DD mice that were NFκB(p50)−/− only in peripheral blood cells (and marrow) versus only in vessel walls (and tissues). This revealed that endothelial TF expression in the NY1DD mouse is highly dependent upon NFκB(p50) in peripheral blood mononuclear cellsbut not in the vessel wall. In confirmation, infusion of post-H/R sickle mouse blood mononuclear cells into naïve NY1DD mice stimulated endothelial TF expression; infusion of such cells from unstimulated sickle mice at ambient air did not stimulate TF expression. We conclude that peripheral blood mononuclear cells indirectly promote endothelial TF expression via a NFκB(p50)-dependent mechanism. This may be relevant to the role of coagulopathy in clinical sickle disease. Keywords sickle; endothelial; tissue factor; NFκB(p50); monocyte; inflammation Address correspondence to Dr. Hebbel, MMC 480, 420 Delaware St. SE, University of Minnesota, Minneapolis, MN 55455. Phone: 612 624 4620, FAX: 612 625 6919, hebbe001@umn.edu. AUTHOR CONTRIBUTIONS RK performed the actual mouse experiments. AS performed all immuno-fluorescence measurements. LCM carried out molecular biology aspects of the project, and supervised breeding strategies which were carried out by FA who bred and genotyped the mice. RJK Jr created, characterized and provided the anti-murine TF antibody. And RPH provided overall supervision, analysis of raw data, data interpretation, creation of all experimental strategies, and wrote the manuscript.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers