Fang Tan

Extracellular hemin crisis triggers acute chest syndrome in sickle mice

The prevention and treatment of acute chest syndrome (ACS) is a major clinical concern in sickle cell disease (SCD). However, the mechanism underlying the pathogenesis of ACS remains elusive. We tested the hypothesis that the hemolysis byproduct hemin elicits events that induce ACS. Infusion of a low dose of hemin caused acute intravascular hemolysis and autoamplification of extracellular hemin in transgenic sickle mice, but not in sickle-trait littermates. The sickle mice developed multiple symptoms typical of ACS and succumbed rapidly. Pharmacologic inhibition of TLR4 and hemopexin replacement therapy prior to hemin infusion protected sickle mice from developing ACS. Replication of the ACS-like phenotype in nonsickle mice revealed that the mechanism of lung injury due to extracellular hemin is independent of SCD. Using genetic and bone marrow chimeric tools, we confirmed that TLR4 expressed in nonhematopoietic vascular tissues mediated this lethal type of acute lung injury. Respiratory failure was averted after the onset of ACS-like symptoms in sickle mice by treating them with recombinant hemopexin. Our results reveal a mechanism that helps to explain the pathogenesis of ACS, and we provide proof of principle for therapeutic strategies to prevent and treat this condition in mice.

Mechanisms of transcriptional regulation and prognostic significance of activated leukocyte cell adhesion molecule in cancer.

BackgroundActivated leukocyte cell adhesion molecule (ALCAM) is implicated in the prognosis of multiple cancers with low level expression associated with metastasis and early death in breast cancer. Despite this significance, mechanisms that regulate ALCAM gene expression and ALCAMs role in adhesion of pre-metastatic circulating tumor cells have not been defined. We studied ALCAM expression in 20 tumor cell lines by real-time PCR, western blot and immunochemistry. Epigenetic alterations of the ALCAM promoter were assessed using methylation-specific PCR and bisulfite sequencing. ALCAMs role in adhesion of tumor cells to the vascular wall was studied in isolated perfused lungs.ResultsA common site for transcription initiation of the ALCAM gene was identified and the ALCAM promoter sequenced. The promoter contains multiple cis-active elements including a functional p65 NF-κB motif, and it harbors an extensive array of CpG residues highly methylated exclusively in ALCAM-negative tumor cells. These CpG residues were modestly demethylated after 5-aza-2-deoxycytidine treatment. Restoration of high-level ALCAM expression using an ALCAM cDNA increased clustering of MDA-MB-435 tumor cells perfused through the pulmonary vasculature of ventilated rat lungs. Anti-ALCAM antibodies reduced the number of intravascular tumor cell clusters.ConclusionOur data suggests that loss of ALCAM expression, due in part to DNA methylation of extensive segments of the promoter, significantly impairs the ability of circulating tumor cells to adhere to each other, and may therefore promote metastasis. These findings offer insight into the mechanisms for down-regulation of ALCAM gene expression in tumor cells, and for the positive prognostic value of high-level ALCAM in breast cancer.

Spatiotemporal Dysfunction of the Vascular Permeability Barrier in Transgenic Mice with Sickle Cell Disease

Sickle cell disease (SCD) is characterized by chronic intravascular hemolysis that generates excess cell-free hemoglobin in the blood circulation. Hemoglobin causes multiple endothelial dysfunctions including increased vascular permeability, impaired reactivity to vasoactive agonists, and increased adhesion of leukocytes to the endothelium. While the adhesive and vasomotor defects of SCD associated with cell-free hemoglobin are well defined, the vascular permeability phenotype remains poorly appreciated. We addressed this issue in two widely used and clinically relevant mouse models of SCD. We discovered that the endothelial barrier is normal in most organs in the young but deteriorates with aging particularly in the lung. Indeed, middle-aged sickle mice developed pulmonary edema revealing for the first time similarities in the chronic permeability phenotypes of the lung in mice and humans with SCD. Intravenous administration of lysed red blood cells into the circulation of sickle mice increased vascular permeability significantly in the lung without impacting permeability in other organs. Thus, increased vascular permeability is an endothelial dysfunction of SCD with the barrier in the lung likely the most vulnerable to acute inflammation.

Global Gene Expression Profiling of Endothelium Exposed to Heme Reveals an Organ-Specific Induction of Cytoprotective Enzymes in Sickle Cell Disease

Background Sickle cell disease (SCD) is characterized by hemolysis, vaso-occlusion and ischemia reperfusion injury. These events cause endothelial dysfunction and vasculopathies in multiple systems. However, the lack of atherosclerotic lesions has led to the idea that there are adaptive mechanisms that protect the endothelium from major vascular insults in SCD patients. The molecular bases for this phenomenon are poorly defined. This study was designed to identify the global profile of genes induced by heme in the endothelium, and assess expression of the heme-inducible cytoprotective enzymes in major organs impacted by SCD. Methods and Findings Total RNA isolated from heme-treated endothelial monolayers was screened with the Affymetrix U133 Plus 2.0 chip, and the microarray data analyzed using multiple bioinformatics software. Hierarchical cluster analysis of significantly differentially expressed genes successfully segregated heme and vehicle-treated endothelium. Validation studies showed that the induction of cytoprotective enzymes by heme was influenced by the origin of endothelial cells, the duration of treatment, as well as the magnitude of induction of individual enzymes. In agreement with these heterogeneities, we found that induction of two major Nrf2-regulated cytoprotective enzymes, heme oxygenase-1 and NAD(P)H:quinone oxidoreductase-1 is organ-specific in two transgenic mouse models of SCD. This data was confirmed in the endothelium of post-mortem lung tissues of SCD patients. Conclusions Individual organ systems induce unique profiles of cytoprotective enzymes to neutralize heme in SCD. Understanding this heterogeneity may help to develop effective therapies to manage vasculopathies of individual systems.

Essential role for ALCAM gene silencing in megakaryocytic differentiation of K562 cells

BackgroundActivated leukocyte cell adhesion molecule (ALCAM/CD166) is expressed by hematopoietic stem cells. However, its role in hematopoietic differentiation has not previously been defined.ResultsIn this study, we show that ALCAM expression is silenced in erythromegakaryocytic progenitor cell lines. In agreement with this finding, the ALCAM promoter is occupied by GATA-1 in vivo, and a cognate motif at -850 inhibited promoter activity in K562 and MEG-01 cells. Gain-of-function studies showed that ALCAM clusters K562 cells in a process that requires PKC. Induction of megakaryocytic differentiation in K562 clones expressing ALCAM activated PKC-δ and triggered apoptosis.ConclusionsThere is a lineage-specific silencing of ALCAM in bi-potential erythromegakaryocytic progenitor cell lines. Marked apoptosis of ALCAM-expressing K562 clones treated with PMA suggests that aberrant ALCAM expression in erythromegakaryocytic progenitors may contribute to megakaryocytopenia.

Cloning of the human activated leukocyte cell adhesion molecule promoter and identification of its tissue-independent transcriptional activation by Sp1

Activated leukocyte cell adhesion molecule (ALCAM) belongs to the immunoglobulin cell adhesion molecule super family. ALCAM is implicated in tumor progression, inflammation, and the differentiation of hematopoietic stem cells. Hitherto, the identity of regulatory DNA elements and cognate transcription factors responsible for ALCAM gene expression remained unknown. In this report, the human ALCAM promoter was cloned and its transcriptional mechanisms elucidated. The promoter is TATA-less and contains multiple GC-boxes. A proximal 650-bp promoter fragment conferred tissue-independent activation, whereas two contiguous regions upstream of this region negatively influenced promoter activity in a tissue-specific manner. The positive regulatory promoter region was mapped to a core 50 base pair sequence containing a conical Sp1 element. Mutation analysis revealed that this element alone or in tandem with elements immediately upstream was required for maximal promoter activity. Chromatin analysis revealed that Sp1 binds exclusively to the canonical binding sequence in vivo, but not to DNA sequence immediately upstream. Finally, we showed that over-expression of Sp1 significantly increased the basal promoter activity. Thus, Sp1 activated the ALCAM promoter in most cells. These findings have important ramifications for unraveling the roles of ALCAM in inflammation and tumorigenesis.

Enhanced down-regulation of ALCAM/CD166 in African-American Breast Cancer.

BackgroundVariation in tumor biology in African-American (AA) and Caucasian (CAU) women with breast cancer is poorly defined. Activated leukocyte cell adhesion molecule (ALCAM) is a bad prognostic factor of breast cancer yet it has never being studied in the AA population. We tested the hypothesis that ALCAM expression would be markedly lower in cases of AA breast cancer when compared to CAU.MethodsCases of breast cancer among AA (n = 78) and CAU (n = 95) women were studied. Immunohistochemical staining was used to semi-quantitatively score ALCAM expression in tumor and adjacent non-tumor breast tissues. Clinico-pathological characteristics including histological type, histological grade, tumor size, lymph node metastasis, estrogen receptor (ER), progesterone receptor (PR), and HER2-neu status were abstracted, and their association with ALCAM expression tested.ResultsUnivariate analysis revealed that the level of ALCAM expression at intercellular junctions of primary tumors correlates with histological grade (AA; p = 0.04, CUA; p = 0.02), ER status (AA; p = 0.0004, CAU; p = 0.0015), PR status (AA; p = 0.002, CUA p = 0.034) and triple-negative tumor status (AA; p = 0.0002, CAU; p = 0.0006,) in both ethnic groups. Multivariate analysis demonstrated that ethnicity contribute significantly to ALCAM expression after accounting for basal-like subtype, age, histological grade, tumor size, and lymph node status. Compared to CAU tumors, the AA are 4 times more likely to have low ALCAM expression (p = 0.003).ConclusionsMarkedly low expression of ALCAM at sites of cell-cell contact in primary breast cancer tumors regardless of differentiation, size and lymph node involvement may contribute to the more aggressive phenotype of breast cancer among AA women.

Original Research: Diametric effects of hypoxia on pathophysiology of sickle cell disease in a murine model

Hypoxia causes erythrocyte sickling in vitro; however, its role in the pathophysiology of sickle cell disease is poorly understood. We report that hypoxia rapidly decreased oxygen saturation in transgenic sickle cell disease mice, but this effect was immediately buffered by a robust ventilatory response. The initial hypoxemia improved steadily throughout the duration of hypoxia without any detectable acute pulmonary adverse effect. Furthermore, the mice suffered acute anemia that ironically was associated with lowering of both plasma hemoglobin and heme. These results were corroborated by increased plasma haptoglobin and hemopexin levels. Markers of ischemic tissue injury increased spatiotemporally following repeated hypoxia exposures. This variation was supported by organ-specific induction of hypoxia-responsive genes. Our results show that hypoxia exerts diametric effects on sickle cell disease by promoting ischemic injury while enhancing the expression of hemolysis scavenger molecules. This phenomenon may help to understand the disparate clinical syndromes associated with hemolysis and vaso-occlusion in sickle cell disease.