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Sickle cell anemia is accompanied by the 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 occurs only after the NY1DD mouse is exposed to hypoxia/reoxygenation (H/R), which actually causes ischemia/reperfusion in the sickle cell disease—but not the normal—mouse model. We tested the hypothesis that the nuclear factor-kappa B (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 (tumor necrosis factor [TNFα], lipopolysaccharide, thioglycollate, and carageenan) given to control mice showed that the inflammatory promotion of TF expression by only pulmonary vein endothelium is not specific to the sickle cell disease model. We bred the NFκB(p50)-/- state into the NY1DD mouse. Combined with marrow transplantation, this allowed the creation of NY1DD mice that were NFκB(p50)-/- only in peripheral blood cells (and marrow) versus only in vessel walls (and tissues). This process revealed that endothelial TF expression in the NY1DD mouse is highly dependent on NFκB(p50) in peripheral blood mononuclear cells—but not in the vessel wall. In confirmation, the infusion of post-H/R sickle mouse blood mononuclear cells into naive NY1DD mice stimulated endothelial TF expression; the infusion of such cells from unstimulated sickle cell disease 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 approach may be relevant to the role of coagulopathy in clinical sickle cell disease.

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.

Purpose: In preclinical models, established molecular determinants of cellular sensitivity to cyclophosphamide, long a mainstay of chemotherapeutic regimens used to treat breast cancers, include the aldehyde dehydrogenases that catalyze the detoxification of this agent, namely, ALDH1A1 and ALDH3A1. As judged by bulk quantification of relevant catalytic activities, as well as of relevant proteins (ELISAs), tissue levels of these enzymes vary widely in primary and metastatic breast malignancies. Thus, interindividual variation in the activity of either of these enzymes in breast cancers could contribute to the wide variation in clinical responses obtained when such regimens are used to treat these malignancies. Direct evidence for this notion was sought in the present investigation. Methods: Cellular levels of ALDH1A1 and ALDH3A1 in 171 repository human breast tumor (122 primary and 49 metastatic) samples were semiquantified using immunocytochemical staining. Clinical responses were retrieved from the archived medical records of each of 48 metastatic breast cancer sample donors, 26 of whom had been treated with a cyclophosphamide-based chemotherapeutic regimen subsequent to tumor sampling and 22 of whom had not. The premise that cellular levels of ALDH1A1 and/or ALDH3A1 predict clinical responses to cyclophosphamide-based chemotherapeutic regimens was submitted to statistical analysis. Results: Confirming an earlier report, ALDH1A1 and ALDH3A1 levels varied widely in both primary and metastatic breast tumor cells. When measurably present, each of the enzymes appeared to be evenly distributed throughout a given tumor cell population. Retrospective analysis indicated that cellular levels of ALDH1A1, but not those of ALDH3A1, were (1) significantly higher in metastatic tumor cells that had survived exposure to cyclophosphamide than in those that had not been exposed to this drug, and (2) significantly higher in metastatic tumors that did not respond (tumor size did not decrease or even increased) to subsequent treatment with cyclophosphamide-based chemotherapeutic regimens than in those that did respond (tumor size decreased) to such regimens. The therapeutic outcome of cyclophosphamide-based chemotherapy corresponded to cellular ALDH1A1 levels in 77% of cases. The frequencies of false-positives (cyclophosphamide-based chemotherapy not effective when a low level of ALDH1A1 predicted it would be) and false-negatives (cyclophosphamide-based chemotherapy effective when a high level of ALDH1A1 predicted it would not be) were 0.00 and 0.43, respectively. Thus, partial or complete responses to cyclophosphamide-based chemotherapy occurred 2.3 times more often when the ALDH1A1 level was low than when it was high. Conclusions: 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.

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; eNOS-Tg) or to have an eNOS knockout state (one eNOS(-/-) animal and several eNOS(+/-) 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.

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; eNOS-Tg) or to have an eNOS knockout state (one eNOS(-/-) animal and several eNOS(+/-) 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.

Clinically, the vascular pathobiology of human sickle cell disease includes an abnormal state of chronic inflammation and activation of the coagulation system. Since these biologies likely underlie development of vascular disease in sickle subjects, they offer attractive targets for novel therapeutics. Similar findings characterize the sickle transgenic mouse, which therefore provides a clinically relevant inflammation model.The authors tested two polyhydroxyphenyl hydroxamic acid derivatives, didox and trimidox, in sickle transgenic mice. Animals were examined by intravital microscopy (cremaster muscle and dorsal skin fold preparations) and by histochemistry before and after transient exposure to hypoxia, with versus without preadministration of study drug. Previous studies have validated the application of hypoxia/reoxygenation to sickle transgenic mice as a disease-relevant model.Animals pretreated with these agents exhibited marked improvements in leukocyte/ endothelial interaction, hemodynamics and vascular stasis, and endothelial tissue factor expression. Thus, these drugs unexpectedly exert powerful inhibition on both the inflammation and coagulation systems.Each of these changes is expected to be therapeutically beneficial in systemic inflammatory disease in general, and in sickle disease in particular. Thus, these novel compounds offer the advantage of having multiple therapeutic benefits in a single agent.

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 (approximately 14% positive), but TF expression is significantly elevated in the moderate and severe mouse models of sickle disease (approximately 29% and approximately 41% positive, respectively). Exposure of the mild sickle mouse to hypoxia for 3 hours, followed by reoxygenation, converted its TF expression phenotype to that of the severe sickle mouse (approximately 36% positive). Pretreatment with lovastatin eliminated excessive expression of TF in the posthypoxic mild sickle mouse (approximately 16% positive) and in the more severe mouse at ambient air (approximately 21% positive). In addition to identifying tissue expression of endothelial TF in the sickle lung, these studies implicate reperfusion injury physiology in its expression and suggest a rationale for use of statins in sickle disease.

In preclinical models, established molecular determinants of cellular sensitivity to cyclophosphamide, long a mainstay of chemotherapeutic regimens used to treat breast cancers, include the aldehyde dehydrogenases that catalyze the detoxification of this agent, namely, ALDH1A1 and ALDH3A1. As judged by bulk quantification of relevant catalytic activities, as well as of relevant proteins (ELISAs), tissue levels of these enzymes vary widely in primary and metastatic breast malignancies. Thus, interindividual variation in the activity of either of these enzymes in breast cancers could contribute to the wide variation in clinical responses obtained when such regimens are used to treat these malignancies. Direct evidence for this notion was sought in the present investigation.Cellular levels of ALDH1A1 and ALDH3A1 in 171 repository human breast tumor (122 primary and 49 metastatic) samples were semiquantified using immunocytochemical staining. Clinical responses were retrieved from the archived medical records of each of 48 metastatic breast cancer sample donors, 26 of whom had been treated with a cyclophosphamide-based chemotherapeutic regimen subsequent to tumor sampling and 22 of whom had not. The premise that cellular levels of ALDH1A1 and/or ALDH3A1 predict clinical responses to cyclophosphamide-based chemotherapeutic regimens was submitted to statistical analysis.Confirming an earlier report, ALDH1A1 and ALDH3A1 levels varied widely in both primary and metastatic breast tumor cells. When measurably present, each of the enzymes appeared to be evenly distributed throughout a given tumor cell population. Retrospective analysis indicated that cellular levels of ALDH1A1, but not those of ALDH3A1, were (1) significantly higher in metastatic tumor cells that had survived exposure to cyclophosphamide than in those that had not been exposed to this drug, and (2) significantly higher in metastatic tumors that did not respond (tumor size did not decrease or even increased) to subsequent treatment with cyclophosphamide-based chemotherapeutic regimens than in those that did respond (tumor size decreased) to such regimens. The therapeutic outcome of cyclophosphamide-based chemotherapy corresponded to cellular ALDH1A1 levels in 77% of cases. The frequencies of false-positives (cyclophosphamide-based chemotherapy not effective when a low level of ALDH1A1 predicted it would be) and false-negatives (cyclophosphamide-based chemotherapy effective when a high level of ALDH1A1 predicted it would not be) were 0.00 and 0.43, respectively. Thus, partial or complete responses to cyclophosphamide-based chemotherapy occurred 2.3 times more often when the ALDH1A1 level was low than when it was high.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.

The mRNA and protein expressions of connexin 26 (Cx26) in rat mammary gland and uterus can be up-regulated during pregnancy as well as by the administration of human CG (hCG). In the present study, we found that the time course and magnitude of Cx26 induction by hCG was different in these two tissues. The molecular mechanism underscoring this difference was therefore investigated. We had previously demonstrated that both Sp1 and Sp3 transcription factors play a functional role in Cx26 expression. By the electrophoretic mobility shift assay, nuclear extracts from both virgin mammary gland and uterus were capable of binding to a labeled oligonucleotide probe that contained the proximal GC box and formed three protein-DNA complexes (C1, C2, and C3). In the mammary gland, pregnancy enhanced the intensity of all three complexes, whereas in the uterine tissue there was a decrease in the C2 and C3 complexes and an emergence of a new major component, C4 complex. In the supershift study, the C1 complex could be supershifted only by an antibody against Sp1, whereas C2, C3, and C4 could all be supershifted by an antibody against Sp3, suggesting a potential presence of Sp3 isoforms of various sizes. We therefore conclude that the basal Sp profiles in virgin mammary gland and uterine tissue are similar. However, in response to pregnancy, the changes in Sp profile are tissue specific and may account for the temporal and quantitative differences between these two tissues in Cx26 induction.

Cell-to-cell communication via gap junctions has played a fundamental role in the orderly development of multicellular organisms. Current methods for measuring this function apply mostly to homotypic cell populations. The newly introduced Fluorescence Activated Cell Sorting (FACS) method, albeit with some limitations, is simple, reliable, and quantitative in measuring the dye transfer via gap junctions in both homotypic and heterotypic cell populations. In the homotypic setting, the result in dye transfer from the FACS method is comparable to the scrape-loading and microinjection methods. Using this FACS method, we observed a decline of cell-to-cell communication in transformed and cancer cells. We also observed a differential degree of communication between two heterotypic cell populations depending on the direction of dye transfer.

Abstract 1048 Peripheral blood monocytes can be pathogenic participants in vascular disease, and they are abnormally activated in sickle patients. As shown earlier, sickle mice have abnormally increased expression of endothelial tissue factor (eTF) in pulmonary veins (PV), expressed as % of PV positive for eTF. For HbS-BERK (eTF 36%, vs 9% for HbA-BERK controls) and S+SAntilles (eTF 28%, vs 7% for C57BL6 controls), abnormal eTF expression is chronic. For NY1DD, however, eTF switches from 7% at ambient air to 23% after exposure to hypoxia/reoxygenation (post-H/R). Having now examined roles of transcription factors Egr-1 and NFkB(p50), we find that H/R triggered expression of eTF in post-H/R NY1DD is: [a] dependent upon both Egr-1 and NFkB(p50) within peripheral blood mononuclear cells (PBMC), and dependent upon Egr-1 butnot NFkB(p50) in vessel wall endothelium. To examine the roles of PBMC and their product TNF in activating eTF expression we used three strategies. [A] First, transfusion (Tx) of PBMC from donor mice (DM) induced eTF in recipient mice (RM) as follows. At-air NY1DD RM exhibited eTF of 7% from Tx of at-air NY1DD DM PBMC, and eTF of 19% from Tx of post-H/R NY1DD DM PBMC. C57BL6 RM exhibited eTF of 10% from Tx of C57BL6 DM PBMC, 31% from Tx of S+SAntilles DM PBMC, and only 15% from Tx of PBMC from S+SAntilles DM also having NFkB(p50)−/−. HbA-BERK RM exhibited eTF of 9% from Tx of HbA-BERK DM PBMC, and 36% from Tx of HbS-BERK DM PBMC. [B] Second, we examined effect of TNF+LT blocker etanercept on PBMC in transfusion experiments. As expected, HbA-BERK RM exhibited eTF 36% from Tx of HbS-BERK PBMC obtained from etanercept-treated (3 mg/kg × 2) DM, and 38% from Tx of HbS-BERK PBMC obtained from DM treated with inactive control TNF-R-scrambled-peptide. On the other hand, HbA-BERK RM pre-treated with etanercept (same dose) vs control TNF-R-scrambled-peptide exhibited eTF of 11% and 33%, respectively from Tx of HbS-BERK DM PBMC. [C] Third, direct TNF blocking pre-treatment of NY1DD mice prevented the eTF increase from subsequent H/R (to 23% for the no pre-treatment controls): eTF of 5% (P=.000013) for pre-treatment with high-etanercept (10 mg/kg × 2), 12% (P=.00059) for pre-treatment with low-etanercept (3 mg/kg × 2), 19% (P=.053) for pre-treatment with IL1-blocker anakinra (10 mg/kg ×2), and 7% (P=.0016) for pretreatment withTNF-specific blocker infliximab (10 mg/kg ×2). For HbS-BERK mice having pre-existing eTF of 36%, extended treatment with etanercept (3 mg/kg, twice weekly × 3) reduced eTF to 27% (P=.026) while those receiving scrambled-peptide remained at 34%. Higher or longer etanercept may be more effective. Other murine treatments have shown us reversal of pre-existing eTF requires extended treatment. Since regulation of TF expression in endothelial cells and blood monocytes is similar, it is likely that these eTF effects would be paralleled by similar effects on monocyte TF expression. Thus, these results suggest that TNF blocking strategies could be helpful in mitigating effects of coagulation activation in the sickle context. Disclosures: No relevant conflicts of interest to declare.

Abstract 2586 Poster Board II-562 The chronic, inflammatory vasculopathy of sickle cell anemia accounts for much of the non-infectious morbidity and mortality of this disease. The pathogenesis of vasculopathy involves multiple sub-biologies (sickling, hemolysis, inflammation, adhesion, coagulation, NO deficiency, stasis, and reperfusion injury). Moreover, these various sub-biologies overlap and transactivate each other. So a single modality approach would very likely fail to be effective. For this reason, effective preventative and interventional therapy for sickle vasculopathy will very likely require true multi-modality therapy, preferably with as few drugs as possible. To this end, we have evaluated the histone deacetylase (HDAC) inhibitors TSA and SAHA for beneficial vascular effects in sickle transgenic mouse models. We used the hBERK1 mouse, which displays abnormal endothelial activation even at ambient air; and we used the NY1DD mouse which only acquires significant endothelial activation upon exposure to hypoxia/reoxygenation. Both drugs significantly inhibited the abnormal endothelial expression of tissue factor, predicting beneficial effects for sickle coagulopathy. Both drugs significantly inhibited abnormal endothelial expression of VCAM1, a molecule involved in both adhesion and inflammation sub-biologies. Both drugs worked in both mouse models. For these benefits, both drugs were effective when given as preventative therapy (NY1DD model) or as longer term therapy (hBERK1 model), although previously-established endothelial activation (e.g., the hBERK1 model) required long term (3 weeks) therapy to observe the most significant benefits. TSA markedly inhibited the vascular stasis in the dorsal skinfold chamber model of hBERK1 mice that is seen after their exposure to H/R; SAHA did so in S+SAntilles mice after H/R; our previous studies have implicated VCAM1 and abnormal cell/cell interaction as the etiology of stasis in this model. Both drugs induced gamma globin and levels of HbF in K562 cells in vitro. Both drugs exhibited activity as iron chelators, as detected by absorption spectra in vitro. Since both drugs are hydroxamic acids, they may promote NO formation by the same mechanism that hydroxyurea is said to work, although we did not test this. We suggest that SAHA, which is already approved for other human clinical use, should be considered for testing in a pilot study to determine drug tolerance and in vivo efficacy in sickle subjects. Disclosures: No relevant conflicts of interest to declare.

Sickle cell anemia is accompanied by biochemical activation of the coagulation system and by clinical thromboses. Therefore, we have focused on the expression of tissue factor (TF), the trigger of the coagulation system, in this disease. Previous studies from our laboratories described abnormal TF expression by monocytes and by circulating endothelial cells in human sickle blood. More recently, we reported (Blood104:840, 2004) that severe-phenotype (BERK) sickle mice abnormally express TF on pulmonary vein endothelium; mild-phenotype (NY1DD) sickle mice are normal in this regard but assume the abnormal phenotype after exposure to hypoxia (3 hr at 8% O2) followed by reoxygenation for 18 hr at ambient air (H/R). Monitoring the % of pulmonary veins positive for TF on triple stained tissue sections (for nuclei, for murine TF, for murine CD31 to identify endothelial cells), we have now evaluated the role of NO in TF expression, employing these two models. In the NY1DD H/R model, NO inhalation (34 ppm) administered during the entire reoxygenation period led to 68±18% reduction (P

By the conventional steroid-binding assay method for receptor, 3% of 1,095 primary breast cancers (or 10.6% of 263 premenopausal tumors) were classified as negative for estrogen receptor (ER), but positive for progesterone receptor (PR). The true ER status in this rare group of tumors was further investigated by the enzyme-immunoassay (EIA) or immunocytochemical (ICA) staining method using monoclonal antibodies H222 and D547. Immunoreactive ER was present in nine ER-/PR+ tumors studied, whereas it was not detectable in nine age-matched ER-/PR- tumors. Immunoreactive ER was also present in 24 ER+ breast cancers studied, and was particularly higher in tumors that were PR+. Measurement of immunoreactive ER by monoclonal antibody method provides certain advantages over the conventional dextran-coated charcoal (DCC) method, especially in ER-/PR+ tumors.