Hormones, age, and sex affect platelet responsiveness in vitro.

BACKGROUND: Female sex confers a survival advantage following severe injury in the setting of trauma-induced coagulopathy, with female platelets having heightened responsiveness likely due to estrogen. The effects of testosterone on platelet biology are unknown, and platelets express both estradiol and androgen receptors on the plasma membrane. We hypothesize testosterone decreases platelet responses in vitro, and there are baseline differences in platelet function and metabolism stratified by sex/age. STUDY DESIGN AND METHODS: Apheresis platelets were collected from: older males (OM) ≥45 years, younger males (YM) <45 years, older females (OF) ≥54 years, and younger females (YF) <54 years, and testosterone and estradiol were measured. Platelets were incubated with testosterone (5.31 ng/ml), estradiol (105 pg/ml) or vehicle and stimulated with buffer, adenosine diphosphate (20 µM), platelet activating factor (2 µM), or thrombin (0.3 U/ml). Aggregation, CD62P surface expression, fibrinogen receptor surface expression, and platelet mitochondrial metabolism were measured. RESULTS: Testosterone significantly inhibited aggregation in OF and OM (p < .05), inhibited CD41a expression in YF, YM, and OM (p < .05), and affected a few of the baseline amounts of CD62P surface expression but not platelet activation to platelet-activating factor and adenosine diphosphate, and variably changed platelet metabolism. DISCUSSION: Platelets have sex- and age-specific aggregation, receptor expression, and metabolism. Testosterone decreases platelet function dependent on the stimulus, age, and sex. Similarly, platelet metabolism has varying responses to sex hormones with baseline metabolic differences dependent upon sex and age.

The Hematopoietic Oxidase NOX2 Regulates Self-Renewal of Leukemic Stem Cells.

The NADPH-dependent oxidase NOX2 is an important effector of immune cell function, and its activity has been linked to oncogenic signaling. Here, we describe a role for NOX2 in leukemia-initiating stem cell populations (LSCs). In a murine model of leukemia, suppression of NOX2 impaired core metabolism, attenuated disease development, and depleted functionally defined LSCs. Transcriptional analysis of purified LSCs revealed that deficiency of NOX2 collapses the self-renewal program and activates inflammatory and myeloid-differentiation-associated programs. Downstream of NOX2, we identified the forkhead transcription factor FOXC1 as a mediator of the phenotype. Notably, suppression of NOX2 or FOXC1 led to marked differentiation of leukemic blasts. In xenotransplantation models of primary human myeloid leukemia, suppression of either NOX2 or FOXC1 significantly attenuated disease development. Collectively, these findings position NOX2 as a critical regulator of malignant hematopoiesis and highlight the clinical potential of inhibiting NOX2 as a means to target LSCs.

Retinoic Acid Is Required for Neural Stem and Progenitor Cell Proliferation in the Adult Hippocampus.

Neural stem and precursor cell (NSPC) proliferation in the rodent adult hippocampus is essential to maintain stem cell populations and produce new neurons. Retinoic acid (RA) signaling is implicated in regulation of adult hippocampal neurogenesis, but its exact role in control of NSPC behavior has not been examined. We show RA signaling in all hippocampal NSPC subtypes and that inhibition of RA synthesis or signaling significantly decreases NSPC proliferation via abrogation of cell-cycle kinetics and cell-cycle regulators. RA signaling controls NSPC proliferation through hypoxia inducible factor-1α (HIF1α), where stabilization of HIF1α concurrent with disruption of RA signaling can prevent NSPC defects. These studies demonstrate a cell-autonomous role for RA signaling in hippocampal NSPCs that substantially broadens RA's function beyond its well-described role in neuronal differentiation.

Col1a1+ perivascular cells in the brain are a source of retinoic acid following stroke.

BACKGROUND: Perivascular stromal cells (PSCs) are a recently identified cell type that comprises a small percentage of the platelet derived growth factor receptor-ß+ cells within the CNS perivascular space. PSCs are activated following injury to the brain or spinal cord, expand in number and contribute to fibrotic scar formation within the injury site. Beyond fibrosis, their high density in the lesion core makes them a potential significant source of signals that act on neural cells adjacent to the lesion site. RESULTS: Our developmental analysis of PSCs, defined by expression of Collagen1a1 in the maturing brain, revealed that PSCs first appear postnatally and may originate from the meninges. PSCs express many of the same markers as meningeal fibroblasts, including expression of the retinoic acid (RA) synthesis proteins Raldh1 and Raldh2. Using a focal brain ischemia injury model to induce PSC activation and expansion, we show a substantial increase in Raldh1+/Raldh2+ PSCs and Raldh1+ activated macrophages in the lesion core. We find that RA levels are significantly elevated in the ischemic hemisphere and induce signaling in astrocytes and neurons in the peri-infarct region. CONCLUSIONS: This study highlights a dual role for activated, non-neural cells where PSCs deposit fibrotic ECM proteins and, along with macrophages, act as a potentially important source of RA, a potent signaling molecule that could influence recovery events in a neuroprotective fashion following brain injury.

N-twist, an evolutionarily conserved bHLH protein expressed in the developing CNS, functions as a transcriptional inhibitor.

Members of the basic helix-loop-helix (bHLH) transcription factor family play an essential role in multiple developmental processes. During neurogenesis, positive and negative regulation by bHLH proteins is essential for proper development. Here we report the identification and initial characterization of the bHLH gene, Neuronal twist (N-twist), named for its neural expression pattern and high sequence homology and physical linkage to the mesodermal inhibitor, M-twist. N-twist is expressed in the developing mouse central nervous system in the midbrain, hindbrain, and neural tube. This neural expression is conserved in invertebrates, as expression of the Drosophila ortholog of N-twist is also restricted to the central nervous system. Like other bHLH family members, N-Twist heterodimerizes with E protein and binds DNA at a consensus bHLH-binding site, the E box. We show that N-Twist inhibits MASH1-dependent transcriptional activation by sequestering E protein in a dominant negative fashion. Thus, these studies support the notion that N-Twist represents a novel negative regulator of neurogenesis.

Drosophila MEF2 is a direct regulator of Actin57B transcription in cardiac, skeletal, and visceral muscle lineages.

To identify regulatory events occurring during myogenesis, we characterized the transcriptional regulation of a Drosophila melanogaster actin gene, Actin 57B. Act57B transcription is first detected in visceral muscle precursors and is detectable in all embryonic muscles by the end of embryogenesis. Through deletion analysis we identified a 595 bp promoter element that was sufficient for high levels of expression in all three muscle lineages. This fragment contained a MEF2 binding site conserved between D. melanogaster and Drosophila virilis which bound MEF2 protein in embryo nuclear extracts. Mutation of the MEF2 site severely reduced promoter activity in embryos, and in Mef2 mutants Act57B expression was severely decreased, demonstrating MEF2 is an essential regulator of Act57B. We also showed that MEF2 likely acts synergistically with factors bound to additional sequences within the 595 bp element. These findings underline the importance of MEF2 in controlling differentiation in all muscle lineages. Our experiments reveal a novel regulatory mechanism for a structural gene where high levels of expression in all embryonic muscles is regulated through a single transcription factor binding site.