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

Activation of the coagulation system is a characteristic feature of sickle cell anemia, which also includes clinical thrombosis. The sickle transgenic mouse abnormally expresses tissue factor (TF) on the pulmonary vein endothelium. Knowing that this aberrancy is stimulated by inflammation, we sought to determine whether nitric oxide (NO) contributes to regulation of endothelial TF expression in the sickle mouse model. We used the NY1DD sickle mouse, which exhibits a low-TF to high-TF phenotype switch on exposure to hypoxia/reoxygenation. Manipulations of NO biology, such as breathing NO or addition of arginine or L-NAME (N-nitro-L-arginine-methyl-ester) to the diet, caused significant modulations of TF expression. This was also seen in hBERK1 sickle mice, which have a different genetic background and already have high-TF even at ambient air. Study of NY1DD animals bred to overexpress endothelial nitric oxide synthase (eNOS; eNOSTg) or to have an eNOS knockout state (one eNOS 2/2 animal and several eNOS 1/2 animals) demonstrated that eNOS modulates endothelial TF expression in vivo by down-regulating it. Thus, the biodeficiency of NO characteristic of patients with sickle cell anemia may heighten risk for activation of the coagulation system. Am. J. Hematol. 85:41-45, 2010. V

Angiogenesis and post-natal vasculogenesis are two processes involved in the formation of new vessels, and both are essential for tumour growth and metastases. We isolated endothelial cells from human blood mononuclear cells by selective culture. These blood outgrowth cells expressed endothelial cell markers and responded correctly to functional assays. To evaluate the potential of blood outgrowth endothelial cells (BOECs) to construct functional vessels in vivo, NOD-SCID mice were implanted with Lewis lung carcinoma cells subcutaneously (s.c.). Blood outgrowth endothelial cells were then injected through the tail vein. Initial distribution of these cells occurred throughout the lung, liver, spleen, and tumour vessels, but they were only found in the spleen, liver, and tumour tissue 48 h after injection. By day 24, they were mainly found in the tumour vasculature. Tumour vessel counts were also increased in mice receiving BOEC injections as compared to saline injections. We engineered BOECs to deliver an angiogenic inhibitor directly to tumour endothelium by transducing them with the gene for human endostatin. These cells maintained an endothelial phenotype and decreased tumour vascularisation and tumour volume in mice. We conclude that BOECs have the potential for tumour-specific delivery of cancer gene therapy.

Elaboration of tumor necrosis factor (TNF) is a very early event in development of ischemia/reperfusion injury pathophysiology. Therefore, TNF may be a prominent mediator of endothelial cell and vascular wall dysfunction in sickle cell anemia, a hypothesis we addressed using NY1DD, S+SAntilles, and SS‐BERK sickle transgenic mice. Transfusion experiments revealed participation of abnormally activated blood monocytes exerting an endothelial activating effect, dependent upon Egr‐1 in both vessel wall and blood cells, and upon NFκB(p50) in a blood cell only. Involvement of TNF was identified by beneficial impact from TNF blockers, etanercept and infliximab, with less benefit from an IL‐1 blocker, anakinra. In therapeutic studies, etanercept ameliorated multiple disturbances of the murine sickle condition: monocyte activation, blood biomarkers of inflammation, low platelet count and Hb, vascular stasis triggered by hypoxia/reoxygenation (but not if triggered by hemin infusion), tissue production of neuro‐inflammatory mediators, endothelial activation (monitored by tissue factor and VCAM‐1 expression), histopathologic liver injury, and three surrogate markers of pulmonary hypertension (perivascular inflammatory aggregates, arteriolar muscularization, and right ventricular mean systolic pressure). In aggregate, these studies identify a prominent—and possibly dominant—role for an abnormal monocyte‐TNF‐endothelial activation axis in the sickle context. Its presence, plus the many benefits of etanercept observed here, argue that pilot testing of TNF blockade should be considered for human sickle cell anemia, a challenging but achievable translational research goal.

BackgroundHealth disparities and the high prevalence of cardiovascular disease continue to be perplexing worldwide health challenges. This study addresses the possibility that genetic differences affecting the biology of the vascular endothelium could be a factor contributing to the increased burden of cardiovascular disease and cancer among African Americans (AA) compared to Caucasian Americans (CA).MethodsFrom self-identified, healthy, 20 to 29-year-old AA (n = 21) and CA (n = 17), we established cultures of blood outgrowth endothelial cells (BOEC) and applied microarray profiling. BOEC have never been exposed to in vivo influences, and their gene expression reflects culture conditions (meticulously controlled) and donor genetics. Significance Analysis of Microarray identified differential expression of single genes. Gene Set Enrichment Analysis examined expression of pre-determined gene sets that survey nine biological systems relevant to endothelial biology.ResultsAt the highly stringent threshold of False Discovery Rate (FDR) = 0, 31 single genes were differentially expressed in AA. PSPH exhibited the greatest fold-change (AA > CA), but this was entirely accounted for by a homolog (PSPHL) hidden within the PSPH probe set. Among other significantly different genes were: for AA > CA, SOS1, AMFR, FGFR3; and for AA < CA, ARVCF, BIN3, EIF4B. Many more (221 transcripts for 204 genes) were differentially expressed at the less stringent threshold of FDR <.05. Using the biological systems approach, we identified shear response biology as being significantly different for AA versus CA, showing an apparent tonic increase of expression (AA > CA) for 46/157 genes within that system.ConclusionsMany of the genes implicated here have substantial roles in endothelial biology. Shear stress response, a critical regulator of endothelial function and vascular homeostasis, may be different between AA and CA. These results potentially have direct implications for the role of endothelial cells in vascular disease (hypertension, stroke) and cancer (via angiogenesis). Also, they are consistent with our over-arching hypothesis that genetic influences stemming from ancestral continent-of-origin could impact upon endothelial cell biology and thereby contribute to disparity of vascular-related disease burden among AA. The method used here could be productively employed to bridge the gap between information from structural genomics (for example, disease association) and cell function and pathophysiology.