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
Given (1) the wide range of ALDH1A1 levels observed in malignant breast tissues, (2) that ALDH1A1 levels in primary breast tumor tissue, as well as those in normal breast tissue, directly reflect ALDH1A1 levels in metastatic breast tumor cells derived therefrom, and (3) the findings reported here, measurement of ALDH1A1 levels in primary breast malignancies and/or normal breast tissue prior to the initiation of chemotherapy is likely to be of value in predicting the therapeutic potential, or lack of potential, of cyclophosphamide and other oxazaphosphorines, e.g. ifosfamide, in the treatment of primary, as well as metastatic, breast cancer, thus providing a rational basis for the design of individualized therapeutic regimens for this disease. Failure to observe the expected inverse relationship between clinical responses to cyclophosphamide-based chemotherapeutic regimens and ALDH3A1 levels was probably because even the highest breast tumor tissue ALDH3A1 level thus far reported appears to be below the threshold level at which ALDH3A1-catalyzed detoxification of oxazaphosphorines becomes pharmacologically meaningful. However, ALDH3A1 levels in certain other malignancies, e.g. those of the alimentary tract and lung, may be of a sufficient magnitude in that regard.
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
The vascular pathobiology of sickle cell anemia involves inflammation, coagulation, vascular stasis, reperfusion injury, iron-based oxidative biochemistry, deficient nitric oxide (NO) bioavailability, and red cell sickling. These disparate pathobiologies intersect and overlap, so it is probable that multimodality therapy will be necessary for this disease. We have, therefore, tested a histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), for efficacy in reducing endothelial activation. We found that pulmonary vascular endothelial VCAM-1 and tissue factor (TF) expression (both are indicators of endothelial activation) are powerfully and significantly inhibited by TSA. This is seen both with pretreatment before the inducing stress of hypoxia/reoxygenation (NY1DD sickle transgenic mouse), and upon longer-term therapy after endothelial activation has already occurred (hBERK1 sickle mouse at ambient air). In addition, TSA prevented vascular stasis in sickle mice, it exhibited activity as an iron chelator, and it induced expression of the antisickling hemoglobin, hemoglobin F. Notably, the TSA analog SAHA (suberoylanilide hydroxaminc acid) that is already approved for human clinical use exhibits the same spectrum of biologic effects as TSA. We suggest that SAHA possibly could provide true, multimodality, salubrious effects for prevention and treatment of the chronic vasculopathy of sickle cell anemia. (Blood. 2010; 115:2483-2490)
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