Grant C. Bullock

Platelet-derived HMGB1 is a critical mediator of thrombosis

Thrombosis and inflammation are intricately linked in several major clinical disorders, including disseminated intravascular coagulation and acute ischemic events. The damage-associated molecular pattern molecule high-mobility group box 1 (HMGB1) is upregulated by activated platelets in multiple inflammatory diseases; however, the contribution of platelet-derived HMGB1 in thrombosis remains unexplored. Here, we generated transgenic mice with platelet-specific ablation of HMGB1 and determined that platelet-derived HMGB1 is a critical mediator of thrombosis. Mice lacking HMGB1 in platelets exhibited increased bleeding times as well as reduced thrombus formation, platelet aggregation, inflammation, and organ damage during experimental trauma/hemorrhagic shock. Platelets were the major source of HMGB1 within thrombi. In trauma patients, HMGB1 expression on the surface of circulating platelets was markedly upregulated. Moreover, evaluation of isolated platelets revealed that HMGB1 is critical for regulating platelet activation, granule secretion, adhesion, and spreading. These effects were mediated via TLR4- and MyD88-dependent recruitment of platelet guanylyl cyclase (GC) toward the plasma membrane, followed by MyD88/GC complex formation and activation of the cGMP-dependent protein kinase I (cGKI). Thus, we establish platelet-derived HMGB1 as an important mediator of thrombosis and identify a HMGB1-driven link between MyD88 and GC/cGKI in platelets. Additionally, these findings suggest a potential therapeutic target for patients sustaining trauma and other inflammatory disorders associated with abnormal coagulation.

Iron control of erythroid development by a novel aconitase-associated regulatory pathway

Human red cell differentiation requires the action of erythropoietin on committed progenitor cells. In iron deficiency, committed erythroid progenitors lose responsiveness to erythropoietin, resulting in hypoplastic anemia. To address the basis for iron regulation of erythropoiesis, we established primary hematopoietic cultures with transferrin saturation levels that restricted erythropoiesis but permitted granulopoiesis and megakaryopoiesis. Experiments in this system identified as a critical regulatory element the aconitases, multifunctional iron-sulfur cluster proteins that metabolize citrate to isocitrate. Iron restriction suppressed mitochondrial and cytosolic aconitase activity in erythroid but not granulocytic or megakaryocytic progenitors. An active site aconitase inhibitor, fluorocitrate, blocked erythroid differentiation in a manner similar to iron deprivation. Exogenous isocitrate abrogated the erythroid iron restriction response in vitro and reversed anemia progression in iron-deprived mice. The mechanism for aconitase regulation of erythropoiesis most probably involves both production of metabolic intermediates and modulation of erythropoietin signaling. One relevant signaling pathway appeared to involve protein kinase Calpha/beta, or possibly protein kinase Cdelta, whose activities were regulated by iron, isocitrate, and erythropoietin.

Cross-talk of GATA-1 and P-TEFb in megakaryocyte differentiation.

The transcription factor GATA-1 participates in programming the differentiation of multiple hematopoietic lineages. In megakaryopoiesis, loss of GATA-1 function produces complex developmental abnormalities and underlies the pathogenesis of megakaryocytic leukemia in Down syndrome. Its distinct functions in megakaryocyte and erythroid maturation remain incompletely understood. In this study, we identified functional and physical interaction of GATA-1 with components of the positive transcriptional elongation factor P-TEFb, a complex containing cyclin T1 and the cyclin-dependent kinase 9 (Cdk9). Megakaryocytic induction was associated with dynamic changes in endogenous P-TEFb composition, including recruitment of GATA-1 and dissociation of HEXIM1, a Cdk9 inhibitor. shRNA knockdowns and pharmacologic inhibition both confirmed contribution of Cdk9 activity to megakaryocytic differentiation. In mice with megakaryocytic GATA-1 deficiency, Cdk9 inhibition produced a fulminant but reversible megakaryoblastic disorder reminiscent of the transient myeloproliferative disorder of Down syndrome. P-TEFb has previously been implicated in promoting elongation of paused RNA polymerase II and in programming hypertrophic differentiation of cardiomyocytes. Our results offer evidence for P-TEFb cross-talk with GATA-1 in megakaryocytic differentiation, a program with parallels to cardiomyocyte hypertrophy.

Isocitrate ameliorates anemia by suppressing the erythroid iron restriction response

The unique sensitivity of early red cell progenitors to iron deprivation, known as the erythroid iron restriction response, serves as a basis for human anemias globally. This response impairs erythropoietin-driven erythropoiesis and underlies erythropoietic repression in iron deficiency anemia. Mechanistically, the erythroid iron restriction response results from inactivation of aconitase enzymes and can be suppressed by providing the aconitase product isocitrate. Recent studies have implicated the erythroid iron restriction response in anemia of chronic disease and inflammation (ACDI), offering new therapeutic avenues for a major clinical problem; however, inflammatory signals may also directly repress erythropoiesis in ACDI. Here, we show that suppression of the erythroid iron restriction response by isocitrate administration corrected anemia and erythropoietic defects in rats with ACDI. In vitro studies demonstrated that erythroid repression by inflammatory signaling is potently modulated by the erythroid iron restriction response in a kinase-dependent pathway involving induction of the erythroid-inhibitory transcription factor PU.1. These results reveal the integration of iron and inflammatory inputs in a therapeutically tractable erythropoietic regulatory circuit.

Aconitase Regulation of Erythropoiesis Correlates with a Novel Licensing Function in Erythropoietin-Induced ERK Signaling

Background Erythroid development requires the action of erythropoietin (EPO) on committed progenitors to match red cell output to demand. In this process, iron acts as a critical cofactor, with iron deficiency blunting EPO-responsiveness of erythroid progenitors. Aconitase enzymes have recently been identified as possible signal integration elements that couple erythropoiesis with iron availability. In the current study, a regulatory role for aconitase during erythropoiesis was ascertained using a direct inhibitory strategy. Methodology/Principal Findings In C57BL/6 mice, infusion of an aconitase active-site inhibitor caused a hypoplastic anemia and suppressed responsiveness to hemolytic challenge. In a murine model of polycythemia vera, aconitase inhibition rapidly normalized red cell counts, but did not perturb other lineages. In primary erythroid progenitor cultures, aconitase inhibition impaired proliferation and maturation but had no effect on viability or ATP levels. This inhibition correlated with a blockade in EPO signal transmission specifically via ERK, with preservation of JAK2-STAT5 and Akt activation. Correspondingly, a physical interaction between ERK and mitochondrial aconitase was identified and found to be sensitive to aconitase inhibition. Conclusions/Significance Direct aconitase inhibition interferes with erythropoiesis in vivo and in vitro, confirming a lineage-selective regulatory role involving its enzymatic activity. This inhibition spares metabolic function but impedes EPO-induced ERK signaling and disturbs a newly identified ERK-aconitase physical interaction. We propose a model in which aconitase functions as a licensing factor in ERK-dependent proliferation and differentiation, thereby providing a regulatory input for iron in EPO-dependent erythropoiesis. Directly targeting aconitase may provide an alternative to phlebotomy in the treatment of polycythemia vera.

Red Blood Cells Store and Release Interleukin-33.

Interleukin-33 (IL-33) is a member of the IL-1 cytokine superfamily that potently drives production of a variety of cytokines and contributes to the pathogenesis of inflammatory diseases. The IL-33 is a nuclear protein and is released from apoptotic or necrotic cells. Serum IL-33 levels are increased in various diseases, such as atopic dermatitis, chronic hepatitis C infection, and asthma. Here, we show that red blood cells (RBCs) are one of the major sources of plasma IL-33. The IL-33 levels are significantly increased in supernatants from lysed RBCs. Plasma IL-33 levels are increased in patients during hemolysis, and plasma IL-33 levels show a positive correlation with degree of hemolysis. The IL-33 protein and messenger RNA levels were detected in the late stages of differentiation in ex vivo primary human erythroid progenitor cell cultures, suggesting that IL-33 is expressed during maturation of RBCs. Furthermore, hemoglobin depleted red cell lysates induced IL-8 expression in human epithelial cells. This effect was attenuated in IL-33 decoy receptor expressing cells and was enhanced in IL-33 receptor expressing cells. These results suggest that erythroid progenitor cells produce IL-33 and circulating RBCs represent a major source of IL-33 that is released upon hemolysis.

Protein kinase D-HDAC5 signaling regulates erythropoiesis and contributes to erythropoietin cross-talk with GATA1

In red cell development, the differentiation program directed by the transcriptional regulator GATA1 requires signaling by the cytokine erythropoietin, but the mechanistic basis for this signaling requirement has remained unknown. Here we show that erythropoietin regulates GATA1 through protein kinase D activation, promoting histone deacetylase 5 (HDAC5) dissociation from GATA1, and subsequent GATA1 acetylation. Mice deficient for HDAC5 show resistance to anemic challenge and altered marrow responsiveness to erythropoietin injections. In ex vivo studies, HDAC5(-/-) progenitors display enhanced entry into and passage through the erythroid lineage, as well as evidence of erythropoietin-independent differentiation. These results reveal a molecular pathway that contributes to cytokine regulation of hematopoietic differentiation and offer a potential mechanism for fine tuning of lineage-restricted transcription factors by lineage-specific cytokines.

Celiac disease refractory to a gluten-free diet?

A 75-year-old woman from an outside hospital was referred because of continued signs and symptoms of celiac disease (gluten-sensitive enteropathy) that persisted despite self-reported adherence to a gluten-free diet. The patient reported excessive gas, bowel distension, a 15-pound weight loss over the past few years, insomnia, and a rash over her lower extremities. The patient had required hospitalizations, intravenous fluids, and continuing therapy with corticosteroids for 6 months. A diagnosis of celiac disease had been made 6 years previously, based on (a) typical gastrointestinal signs and symptoms with negative stool cultures and Clostridium difficile toxin assay, (b) positive serology for celiac disease, (c) unremarkable colonoscopy with normal random biopsy results, and (d) small-bowel biopsy results showing evidence of villous blunting with increased chronic inflammatory cells. At that time, the patient’s laboratory results included antigliadin antibody (AGA) IgG 0.8 AU (<10 AU), anti-AGA IgA 1.1 AU (<5 AU), anti–tissue transglutaminase (tTG) IgA 9.2 AU (<4 AU), and normal total IgA and IgA antiendomysial antibody (EMA) values. A computed tomographic scan was negative for lymphoma, and an upper gastrointestinal series and small-bowel follow-through barium x-ray were normal. Endoscopic biopsy results obtained during the previous 2 years showed continued villous atrophy with intraepithelial lymphocytes. Shortly before the patient’s referral, repeat biopsies showed villous blunting with increased chronic inflammation, findings confirmed by a gastrointestinal pathologist at our institution. The patient, a pleasant, frail-looking, elderly woman in no acute distress, was retired and married with 2 adult children. She denied smoking and alcohol use and had no family history of celiac disease, liver disease, or colon cancer. Her medical history was remarkable for placement of a carotid artery stent 5 years earlier. Physical examination was unremarkable except for the presence of a maculopapular rash inconsistent with dermatitis herpetiformis and with dependent distribution over the lower …

Nitric Oxide–Independent Soluble Guanylate Cyclase Activation Improves Vascular Function and Cardiac Remodeling in Sickle Cell Disease

&NA; Sickle cell disease (SCD) is associated with intravascular hemolysis and oxidative inhibition of nitric oxide (NO) signaling. BAY 54‐6544 is a small‐molecule activator of oxidized soluble guanylate cyclase (sGC), which, unlike endogenous NO and the sGC stimulator, BAY 41‐8543, preferentially binds and activates heme‐free, NO‐insensitive sGC to restore enzymatic cGMP production. We tested orally delivered sGC activator, BAY 54‐6544 (17 mg/kg/d), sGC stimulator, BAY 41‐8543, sildenafil, and placebo for 4‐12 weeks in the Berkeley transgenic mouse model of SCD (BERK‐SCD) and their hemizygous (Hemi) littermate controls (BERK‐Hemi). Right ventricular (RV) maximum systolic pressure (RVmaxSP) was measured using micro right‐heart catheterization. RV hypertrophy (RVH) was determined using Fultons index and RV corrected weight (ratio of RV to tibia). Pulmonary artery vasoreactivity was tested for endothelium‐dependent and ‐independent vessel relaxation. Right‐heart catheterization revealed higher RVmaxSP and RVH in BERK‐SCD versus BERK‐Hemi, which worsened with age. Treatment with the sGC activator more effectively lowered RVmaxSP and RVH, with 90‐day treatment delivering superior results, when compared with other treatments and placebo groups. In myography experiments, acetylcholine‐induced (endothelium‐dependent) and sodium‐nitroprusside‐induced (endothelium‐independent NO donor) relaxation of the pulmonary artery harvested from placebo‐treated BERK‐SCD was impaired relative to BERK‐Hemi but improved after therapy with sGC activator. By contrast, no significant effect for sGC stimulator or sildenafil was observed in BERK‐SCD. These findings suggest that sGC is oxidized in the pulmonary arteries of transgenic SCD mice, leading to blunted responses to NO, and that the sGC activator, BAY 54‐6544, may represent a novel therapy for SCD‐associated pulmonary arterial hypertension and cardiac remodeling.

Posttransplantation lymphoproliferative disease involving the pituitary gland

Posttransplantation lymphoproliferative disorders (PTLD) are heterogeneous lesions with variable morphology, immunophenotype, and molecular characteristics. Multiple distinct primary lesions can occur in PTLD, rarely with both B-cell and T-cell characteristics. Lesions can involve both grafted organs and other sites; however, PTLD involving the pituitary gland has not been previously reported. We describe a patient who developed Epstein-Barr virus-negative PTLD 13 years posttransplantation involving the terminal ileum and pituitary, which was simultaneously involved by a pituitary adenoma. Immunohistochemistry of the pituitary lesion showed expression of CD79a, CD3, and CD7 with clonal rearrangements of both T-cell receptor gamma chain (TRG@) and immunoglobulin heavy chain (IGH@) genes. The terminal ileal lesion was immunophenotypically and molecularly distinct. This is the first report of pituitary PTLD and illustrates the potentially complex nature of PTLD.