ALBERT

Description: Sickle cell disease is a common hemolytic disorder with a broad range of complications, including vaso-occlusive episodes, acute chest syndrome (ACS), pain, and stroke. Heme oxygenase-1 (gene HMOX1; protein HO-1) is the inducible, rate-limiting enzyme in the catabolism of heme and might attenuate the severity of outcomes from vaso-occlusive and hemolytic crises. A (GT)n dinucleotide repeat located in the promoter region of the HMOX1 gene is highly polymorphic, with long repeat lengths linked to decreased activity and inducibility. We examined this polymorphism to test the hypothesis that short alleles are associated with a decreased risk of adverse outcomes (hospitalization for pain or ACS) among a cohort of 942 children with sickle cell disease. Allele lengths varied from 13 to 45 repeats and showed a trimodal distribution. Compared with children with longer allele lengths, children with 2 shorter alleles (4%; ≤ 25 repeats) had lower rates of hospitalization for ACS (incidence rate ratio 0.28, 95% confidence interval, 0.10-0.81), after adjusting for sex, age, asthma, percentage of fetal hemoglobin, and α-globin gene deletion. No relationship was identified between allele lengths and pain rate. We provide evidence that genetic variation in HMOX1 is associated with decreased rates of hospitalization for ACS, but not pain. This study is registered at www.clinicaltrials.gov as #NCT00072761.

Description: Activated leukocyte cell adhesion molecule (ALCAM/CD166) is a member of the immunoglobulin super-family. It is expressed on the surfaces of activated monocytes, dendritic cells and macrophages. These immune cells use ALCAM through homotypic and heterotypic adhesions to control multiple stages in the inflammatory response. Indeed, anti-ALCAM antibodies and recombinant soluble ALCAM significantly inhibit monocyte transendothelial migration, stabilization of the immunological synapse and dendritic cell-mediated T-lymphocyte proliferation. Despite this significance, there is currently no understanding of how the human ALCAM gene is regulated. In this study, we identified the mechanisms for transcription, basal transcriptional activation and immunosuppressive silencing of the ALCAM gene. A common site for transcription of the ALCAM gene was identified 350 base pairs (bp) upstream from the translational start site. Multiple truncated fragments of the ALCAM promoter was cloned from genomic DNA and sub-cloned upstream of a promoterless luciferase vector. A proximal 650-bp promoter sequence conferred tissue-independent activation in hematopoietic, epithelial and endothelial cells. A canonical Sp1 binding sequence at −550 upstream of the translational start site was mapped within this proximal positive regulatory promoter region. Site-directed mutagenesis revealed this sequence was essential for optimum ALCAM promoter activity. Importantly, Sp1 occupied the cognate sequence in vivo as determined by chromatin immunoprecipitation assays. Over-expression of Sp1 significantly increased ALCAM promoter activity whereas a control expression vector had no impact. DNA sequences in the interval −600 to −800 negatively influenced promoter activity in a tissue-specific manner. This region contained a putative binding sequence for the aryl hydrocarbon receptor (Ahr), which highlighted ALCAM as a potential target of the immunosuppressing ligand dioxin. This hypothesis was tested by examination of whether ALCAM activation is blocked by 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) in monocytes differentiating into macrophages and dendritic cells. Expression of ALCAM was increased 3–5-fold in HL-60 and THP-1 monocytes treated with the differentiating agent phorbol 12-myristate 13-acetate. TCDD dose dependently blocked this activation, indeed, the highest concentration of TCDD (25 nM) used in this study completely blocked ALCAM activation in both monocytic cells. In conclusion, we have unveiled for the first time, the molecular basis for transcription and basal trans-activation of the human ALCAM gene, and identified the Ahr-pathway as a powerful silencer of ALCAM gene activation. Further studies of the ALCAM promoter, may clarify how this gene is up-regulated as part of the inflammatory response, and how it is silenced by immunotoxins. Heterologous expression of ALCAM may be a potential strategy to mitigate the immunosuppressive effects of dioxins and polycyclic aromatic hydrocarbons.

Description: Abstract 4836 Sickle cell disease (SCD) is a chronic hemolytic and inflammatory disorder characterized by repeated episodes of vaso-occlusion and hemolysis, resulting in oxidative stress and endothelial dysfunction. We have recently demonstrated that the heme scavenging capacity in SCD is severely impaired, highlighting the danger posed by excess heme in this disorder. Paradoxically, heme induces expression of several cyto-protective enzymes including the modifier subunit of glutamate cysteine ligase (GCLM), the rate-limiting enzyme in glutathione (GSH) synthesis, which is a crucial antioxidant in the lung. While the induction of cytoprotective enzymes is thought to attenuate the deleterious effects of heme in SCD the somatic origin of this protection has not previously been defined. Using transgenic mouse models we show for the first time that the level of GCLM in the sickle lung is markedly up-regulated due primarily to enhanced expression of the enzyme in the epithelium and blood mononuclear cells, but not in the endothelium. Based on these findings, we tested the hypothesis that leukocyte-derived GCLM was sufficient to protect the sickle lung from oxidative stress. Thus, bone marrow chimeric SCD mice with GCLM deficiency were generated by transplanting bone marrow from Berkeley SCD transgenic mice into GCLM null mice recipients. We confirmed that the chimeric GCLM-null-SCD mice had a SCD phenotype as determined by 〉95% engraftment of donor white blood cells, reticulocyte counts, urine osmolality and hemoglobin gel electrophoresis. Whole lung GCLM and total GSH levels in the chimeric mice were identical to the levels in the wild-type SCD mice. Moreover, lung function, as determined by oxygen saturation and breath rate, were identical in the two mouse strains. These results show that loss of GCLM expression in resident lung cells does not compromise production of GSH or the function of the lung in SCD. Disclosures: Ofori-Acquah: Emory University: Patents & Royalties.

Description: Nuclear factor erythroid-2 related factor 2 (Nrf2) is a transcription factor that regulates the cellular defense mechanism by mediating a coordinate induction of cytoprotective antioxidant responsive element-driven genes. Nrf2 agonists augment fetal hemoglobin expression in hematopoietic progenitors and have emerged as a new class of drugs for therapeutic induction of fetal hemoglobin in sickle cell anemia (SCA). However, the cytoprotective effect of Nrf2 on the pathobiology of SCA has not been previously defined. To investigate the role and mechanism of Nrf2 in SCA independent of globin gene modulation, we generated chimeric mice with disruption of Nrf2 in non-hematopoietic tissues. A total of twenty-six Nrf2-/- mice were transplanted with bone marrow harvested from the Berkeley sickle and hemizygous (Hemi) mice and the Townes sickle (SS) and control (AA) mice. All sickle/Nrf2-null chimeras (SSNHNrf2-/-; n=13) developed classical hematological and intravascular hemolysis features of SCA after eight weeks of transplantation. Despite the presence of erythrocyte Nrf2, SSNHNrf2-/- chimeras developed more severe intravascular hemolysis than SS wild-type (SSWT) donor-litter-mates. The concentration of plasma cell-free Hb (p

Description: The histone deacetylase inhibitors (HDA-CIs) butyrate and trichostatin A activate γ-globin expression via a p38 mitogen-activating protein kinase (MAPK)-dependent mechanism. We hypothesized that down-stream effectors of p38 MAPK, namely activating transcription factor-2 (ATF-2) and cyclic AMP response element (CRE) binding protein (CREB), are intimately involved in fetal hemoglobin induction by these agents. In this study, we observed increased ATF-2 and CREB1 phosphorylation mediated by the HDACIs in K562 cells, in conjunction with histone H4 hyperacetylation. Moreover, enhanced DNA-protein interactions occurred in the CRE in the Gγ-globin promoter (G-CRE) in vitro after drug treatments; subsequent chromatin immunoprecipitation assay confirmed ATF-2 and CREB1 binding to the G-CRE in vivo. Enforced expression of ATF-2 and CREB produced Gγ-promoter trans-activation which was abolished by a 2-base pair mutation in the putative G-CRE. The data presented herein demonstrate that γ-gene induction by butyrate and trichostatin A involves ATF-2 and CREB1 activation via p38 MAPK signaling.

Description: Abstract 2135 Circulating heme is scavenged by multiple plasma proteins and delivered to the liver for degradation. We have recently demonstrated that heme scavenging is variably impaired in transgenic mice with sickle cell disease (SCD). In this study, we tested the hypothesis that excess protein-free plasma heme (PFPH) in the blood circulation, and excess scavenged protein-bound plasma heme (PBPH) destined for degradation cause different types of organ damage in SCD. Transgenic mice expressing exclusively human sickle hemoglobin (SS) were intravenously injected with a dose of heme (25 micromoles/kg) to elevate PBPH only, or with a dose (70 micromoles/kg) sufficient to raise PFPH. In agreement with our previous findings, PFPH was associated with severe lung injury and 100% lethality within 2 hours. This phenomenon occurred independent of any liver involvement. Modest elevation of circulating heme sufficient to raise PBPH only, increased alanine aminotransferase and aspartate aminotranferase 3- to 4-fold (P

Description: Abstract 944 Acute chest syndrome (ACS) is the leading cause of death among patients with sickle cell disease (SCD). It is a process of devastating acute lung injury that evolves from multiple exacerbating events including vaso-occlusive pain crises, infection and fat emboli. ACS results in pulmonary infiltration, hypoxemia, and occlusions in the pulmonary microcirculation. Hitherto, experimental models of ACS have been lacking, and molecular targets of therapy remain to be identified. Clinical studies indicate that most patients diagnosed with ACS hemolyse during the acute phase of the syndrome, which highlights a role for circulating heme/hemin in this process. Since the deleterious effects of hemin are defined by increased vascular permeability, we tested the hypothesis that acute elevation of circulating hemin would increase pulmonary microvascular leakage sufficiently to trigger ACS. Adult transgenic mice expressing exclusively human sickle hemoglobin (Hb SS), and control Hb AS and Hb AA mice were intravenously injected with hemin (70 micromoles/kg body weight), and cardiopulmonary function assessed in real-time using a mouse pulse oximeter. Arterial oxygen saturation (SpO2) in the SS mice reduced significantly (p = 0.02) to 84.1 ± 5.6 % from a normal baseline value of 98.6 ± 0.3 %, within 25 minutes of administration of i.v. hemin, while SpO2 in control AS and AA mice remained unchanged. Consistent with changes in cardiopulmonary function, all the SS mice (n=14) succumbed to hemin, within 2 hours, while all control AS and AA mice survived, and remained alive several weeks after the experiment (log-rank survival test, p=

Description: Migration of endothelial cells as a sheet in fully differentiated blood vessels is essential for reducing vascular permeability during wound healing. Indeed, loss of collective endothelial sheet migration contributes to increased vascular permeability in tumor angiogenesis and several vascular proliferative disorders. Despite this significance, mechanisms responsible for keeping migrating endothelial cells in a monolayer, sheet or tube are poorly understood. To unravel the basis for collective endothelial cell migration, we used time-lapse video microscopy to study early events of wound closure in confluent monolayers of primary microvascular endothelial cells in a live cell chamber. Immediately after wounding (0– 20 min), endothelial cells at the margin of the wound (marginal cells) retracted away from the wounded area and showed no visible lamellipodia extensions. The next phase of early wound healing (20 min – 6 hours) revealed extensive lamellipodia formation and migration of marginal cells into the wounded region. Remarkably, sub-marginal endothelial cells that were several microns away from the wound edge protruded lamellipodia that formed dynamic cell-cell contacts with the substratum of marginal cells at the wound edge. In several instances sub-marginal cells physically and coordinately pulled back endothelial cells at wound edge to maintain regularity of the endothelial sheet front. Cell-tracking measurements revealed autonomous and yet coordinated migration of marginal and sub-marginal endothelial cells culminating in net protrusion of the endothelial sheet into the wound. This study provides in real-time evidence of retraction of endothelial cells at the wound edge by several microns prior to the initiation of forward migration. In addition, we show for the first time that endothelial cells several microns away from the wound edge actively participate in sheet migration through the extention of lamellipodia into the substratum of cells at the wound edge. These findings highlight an important role in endothelial sheet migration for the Rho family of GTPases given their intimate control of cell retraction and lamelipodia extensions. Future studies will directly evaluate the influence of Rac, Cdc42 and RhoA in retraction of marginal cells and formation of lamellipodia by sub-marginal cells in endothelial sheet migration.

Description: Activated leukocyte cell adhesion molecule (ALCAM/CD166) is a member of the immunoglobulin cell adhesion super family, which has been implicated in diverse physiological and pathophysiological events involving cell migration. Hitherto, ALCAM’s role in inflammation has not been determined. In this study, we show ALCAM is involved in controlling migration of mononuclear leukocytes across the pulmonary endothelium. We demonstrated that ALCAM is localized at intercellular junctions in pulmonary microvascular endothelial cells in vitro and in vivo. ALCAM co-localized with multiple adherens junction molecules including cadherins, catenins and Dlg, as determined by confocal microscopy, and these observations were confirmed by co-immunoprecipitation and co-distribution assays. Treatment of endothelial cultures with EGTA and cytochalasin D translocated ALCAM from intercellular junctions to the cytosol indicating a requirement for homotypic cadherin adhesion and an intact endothelial cytoskeleton for maintaining ALCAM at endothelial cell junctions. Collectively, these data supports the conclusion that ALCAM contributes to the adherens junction complex in endothelial cells. To determine ALCAM’s role in leukocyte-endothelial cell interactions, adult Sprague Dawley rats were intratracheally instilled with macrophage inflammatory protein-1, and this treatment caused acute expression of ALCAM exclusively in newly recruited mononuclear but not polymorphonuclear leukocytes in the alveolar airway. Given that no ALCAM reactivity was observed in peripheral blood leukocytes, we concluded ALCAM is activated as part of the phenotypic switch by mononuclear leukocytes transitioning from circulation to interstitial tissue compartments. To determine the physiological relevance of this finding we examined whether ALCAM was required for transendothelial migration using monocyte chemoattractant protein 1 (MCP-1). MCP-1 dose- and time-dependently increased the number of transmigrated THP-1 monocytes across pulmonary microvascular endothelial monolayers. Recombinant soluble ALCAM dose-dependently reduced the number of transmigrated THP-1 monocytes, whereas in control experiments recombinant soluble vascular endothelial cadherin had no effect on transmigration. This study shows for the first time that ALCAM is located at endothelial cell junctions where it is intimately involved in controlling the number of monocytes that pass through endothelial barriers. ALCAM may therefore play an essential role in the response to inflammation by enhancing recruitment of mononuclear leukocytes by inflamed tissues.