Proliferation Of Bone Marrow Progenitors Research Articles

Endothelial Damage in JAK2V617F Myeloproliferative Neoplasms with Splanchnic Vein Thrombosis.

JAK2V617F-mutated myeloproliferative neoplasms (MPN) exhibit abnormal proliferation of bone marrow progenitors and increased risk of thrombosis, specifically in splanchnic veins (SVT). The contribution of the endothelium to the development of the prothrombotic phenotype was explored. Plasma and serum samples from JAK2V617F MPN patients with (n=26) or without (n=7) thrombotic debut and different treatments, were obtained (n=33). Cultured endothelial cells (ECs) were exposed to serum samples from these patients and from healthy donors as controls. Changes in markers of inflammation (VCAM-1, ICAM-1), cell permeability (VE-cadherin), production of VWF, extracellular matrix (ECM) reactivity, and activation of intracellular signaling pathways related to stress, proliferation, inflammation (Akt, p44/42, IkBa), and JAK2/STAT3 pathway, were assessed by immunofluorescence, flow adhesion, SDS-PAGE and immunoblot. Additionally, circulating markers of endothelial activation and damage (VWF, sVCAM-1, sTNFRI, thrombomodulin, angiopoietin-2, a2-antiplasmin activity, PAI-1) were evaluated in Patients' plasma. The in vitro studies showed that EC exposure to MPN thrombotic patients' sera resulted in increased VCAM-1 and ICAM-1, and reduced VE-cadherin expression (p<0.05) at the cell surface. Production and release of VWF to the ECM were higher (p<0.05), with increased platelet adhesion after perfusing whole blood, being more noticeable in response to sera from non-treated patients. Furthermore, intracellular activation of Akt, p44/42, IkBa and JAK2/STAT3 was observed. Moreover, plasma levels of VWF, TNF-R1, VCAM-1, thrombomodulin, and angiopoietin-2 were higher in JAK2V617F+ MPN patients with thrombosis. The present findings suggest that circulating factors in MPNs with SVT debut induce endothelial proinflammatory and prothrombotic phenotypes, which are modulated in vitro with MPN treatment.

Abstract 15830: Pharmacological Inhibition of Lysophosphatidic Acid Reduces Inflammation and Enhances Myocardial Recovery After Acute Infarction in Diet-Induced Obese Mice

Introduction: Obesity is approaching epidemic status in the United States and is strongly associated with a higher risk of heart failure after myocardial infarction (MI), a major cause of morbidity and mortality in obese individuals. However, the mechanisms underlying obesity-associated heart failure are poorly understood. Lysophosphatidic acid (LPA), produced by autotaxin (ATX), is highly expressed in adipose tissue with increased levels in obesity and has been shown to mediate post-MI inflammation. Methods: 3-week aged male C57BL/6J mice were randomly assigned to low-fat (LFD-10%) or high-fat (HFD-60%) diet based on the percentage of calories from fat. HFD-fed mice were randomly assigned to receive the specific ATX inhibitor, PF-8380 at 10 mg/kg twice daily. 2 months after initiating the diet, mice were randomized to undergo MI surgery (permanent ligation of the left anterior descending artery) or sham surgery. Results: MI was associated with an increased number of circulating inflammatory monocytes (CD45 + /Ly6C + /CD115 + ), as well as cardiac total and pro-inflammatory macrophages (CD45 + /F4-80 + /CD11b + /CD86 + ), as assessed by flow cytometry (Fig. 1A). This effect was exacerbated in HFD-fed mice but significantly attenuated in HFD+PF8380 treated mice with effective ATX inhibition. Changes in circulating and cardiac immune cells were reflective of increased proliferation of bone marrow progenitors, a phenomenon that was blunted by ATX inhibition (Fig. 1B). Moreover, HFD was associated with larger scar size (Fig. 1C) and worse cardiac functional recovery (Fig. 1D) 30 days after MI. The obesity-asscoiated heart failure could be rescued by ATX inhibition. Conclusion: ATX/LPA plays an important role in modulating inflammation and could be a therapeutic target for obesity-related coronary heart diseases.

Comparable Effects of the Androgen Derivatives Danazol, Oxymetholone and Nandrolone on Telomerase Activity in Human Primary Hematopoietic Cells from Patients with Dyskeratosis Congenita

Dyskeratosis congenita (DKC) is a rare inherited disease of impaired telomere maintenance that progressively leads to multi-organ failure, including the bone marrow. By enhancing telomerase activity, androgen derivatives (ADs) are a potential therapeutic option able to re-elongate previously shortened telomeres. Danazol, oxymetholone, and nandrolone are ADs most frequently used to treat DKC. However, no direct in vitro analyses comparing the efficacy of these ADs have been conducted so far. We therefore treated mononuclear cells derived from peripheral blood and bone marrow of four patients with mutations in telomerase reverse transcriptase (TERT, n = 1),in the telomerase RNA component (TERC, n = 2) and in dyskerin pseudouridine synthase 1 (DKC1, n = 1) and found no substantial differences in the activity of these three agents in patients with TERC/TERT mutations. All AD studied produced comparable improvements of proliferation rates as well as degrees of telomere elongation. Increased TERT expression levels were shown with danazol and oxymetholone. The beneficial effects of all ADs on proliferation of bone marrow progenitors could be reversed by tamoxifen, an estrogen antagonist abolishing estrogen receptor-mediated TERT expression, thereby underscoring the involvement of TERT in AD mechanism of action. In conclusion, no significant differences in the ability to functionally enhance telomerase activity could be observed for the three AD studied in vitro. Physicians therefore might choose treatment based on patients’ individual co-morbidities, e.g., pre-existing liver disease and expected side-effects.

ERK Signaling Is Essential for Macrophage Development.

Macrophages depend on colony stimulating factor 1 (also known as M-CSF) for their growth and differentiation, but the requirements for intracellular signals that lead to macrophage differentiation and function remain unclear. M-CSF is known to activate ERK1 and ERK2, but the importance of this signaling pathway in macrophage development is unknown. In these studies, we characterized a novel model of Erk1 -/- Erk2 flox/flox Lyz2 Cre/Cre mice in which the ERK2 isoform is deleted from macrophages in the background of global ERK1 deficiency. Cultures of M-CSF-stimulated bone marrow precursors from these mice yielded reduced numbers of macrophages. Whereas macrophages developing from M-CSF-stimulated bone marrow of Erk2 flox/flox Lyz2 Cre/Cre mice showed essentially complete loss of ERK2 expression, the reduced number of macrophages that develop from Erk1 -/- Erk2 flox/flox Lyz2 Cre/Cre bone marrow show retention of ERK2 expression, indicating selective outgrowth of a small proportion of precursors in which Cre-mediated deletion failed to occur. The bone marrow of Erk1 -/- Erk2 flox/flox Lyz2 Cre/Cre mice was enriched for CD11b+ myeloid cells, CD11bhi Gr-1hi neutrophils, Lin- c-Kit+ Sca–1+ hematopoietic stem cells, and Lin- c-Kit+ CD34+ CD16/32+ granulocyte-macrophage progenitors. Culture of bone marrow Lin- cells under myeloid-stimulating conditions yielded reduced numbers of monocytes. Collectively, these data indicate that the defect in production of macrophages is not due to a reduced number of progenitors, but rather due to reduced ability of progenitors to proliferate and produce macrophages in response to M-CSF-triggered ERK signaling. Macrophages from Erk1 -/- Erk2 flox/flox Lyz2 Cre/Cre bone marrow showed reduced induction of M-CSF-regulated genes that depend on the ERK pathway for their expression. These data demonstrate that ERK1/ERK2 play a critical role in driving M-CSF-dependent proliferation of bone marrow progenitors for production of macrophages.

Gene Trap Mice Reveal an Essential Function of Dual Specificity Phosphatase Dusp16/MKP-7 in Perinatal Survival and Regulation of Toll-like Receptor (TLR)-induced Cytokine Production

MAPK activity is negatively regulated by members of the dual specificity phosphatase (Dusp) family, which differ in expression, substrate specificity, and subcellular localization. Here, we investigated the function of Dusp16/MKP-7 in the innate immune system. The Dusp16 isoforms A1 and B1 were inducibly expressed in macrophages and dendritic cells following Toll-like receptor stimulation. A gene trap approach was used to generate Dusp16-deficient mice. Homozygous Dusp16tp/tp mice developed without gross abnormalities but died perinatally. Fetal liver cells from Dusp16tp/tp embryos efficiently reconstituted the lymphoid and myeloid compartments with Dusp16-deficient hematopoietic cells. However, GM-CSF-induced proliferation of bone marrow progenitors in vitro was impaired in the absence of Dusp16. In vivo challenge with Escherichia coli LPS triggered higher production of IL-12p40 in mice with a Dusp16-deficient immune system. In vitro, Dusp16-deficient macrophages, but not dendritic cells, selectively overexpressed a subset of TLR-induced genes, including the cytokine IL-12. Dusp16-deficient fibroblasts showed enhanced activation of p38 and JNK MAPKs. In macrophages, pharmacological inhibition and siRNA knockdown of JNK1/2 normalized IL-12p40 secretion. Production of IL-10 and its inhibitory effect on IL-12 production were unaltered in Dusp16tp/tp macrophages. Altogether, the Dusp16 gene trap mouse model identifies an essential role in perinatal survival and reveals selective control of differentiation and cytokine production of myeloid cells by the MAPK phosphatase Dusp16.

Vasoactive intestinal peptide (VIP) inhibits the proliferation of bone marrow progenitors through the VPAC1 receptor.

The cellular and molecular mechanisms of hematopoietic stimulation have been studied. However, an understanding of negative effects in the hematopoietic system remains elusive. To this end, we studied the effects of vasoactive intestinal peptide (VIP) on bone marrow (BM) progenitors. Different BM cell subsets were used to perform clonogenic assay for granulocytic (CFU-GM) or erythroid (BFU-E and CFU-E) progenitors with 10(-7)-10(-13) M VIP. The relevant receptor was verified with specific antagonists, or agonists, semi-quantitative RT-PCR, and chemical cross-linking studies with stromal membranes. Assays performed with unfractionated mononuclear cells and enriched CD34(+) cells showed dose-dependent inhibition on BM progenitors with significant inhibition up to 10(-10) M. Nylon wool separated cells, which depleted stroma, reversed the inhibitory effects of VIP between 10 and 20%. Combined experimental evaluation indicated that the effects of VIP on BM functions are mediated through the type 1 receptor (VPAC1). VIP induced the production of TGF-beta and TNF-alpha in BM mononuclear cells and stroma. These cytokines are partly involved in reversing the suppressive effects of VIP on CFU-GM. The effect of VIP on BM progenitors could be mediated through direct and indirect mechanism. Direct effects were evident by the suppressive effects of VIP on clonogenic assays with highly purified CD34(+) cells. Indirect effects were mediated through putative functions of the stromal cells and the production of TGF-beta and TNF-alpha.

Induction of preprotachykinin-I and neurokinin-1 by adrenocorticotropin and prolactin. Implication for neuroendocrine-immune-hematopoietic axis

We studied the complex interactions within the neuroendocrine-immune-hematopoietic axis by determining a possible link among ACTH, PRL, PPT-I and the receptors for its peptides, NK-1 and NK-2. Indeed, ACTH and PRL induced the expression of PPT-I and NK-1 in human bone marrow stroma with no effect on NK-2. Consistent with a role for PPT-I in regulating the development of myeloid and erythroid progenitors, we found that ACTH and PRL, through NK-1 stimulated the proliferation of both types of progenitors. Induction of PPT-I was regulated at the transcriptional and post-transcriptional levels. The results showed that ACTH and PRL stimulated the proliferation of bone marrow progenitors, partly through PPT-I and NK-1 induction.

Inherited Complete Deficiency of 20-Kilodalton Homologous Restriction Factor (CD59) as a Cause of Paroxysmal Nocturnal Hemoglobinuria

PAROXYSMAL nocturnal hemoglobinuria is a rare acquired disease caused by an unusual susceptibility of erythrocytes to the lytic action of complement. The abnormal erythrocytes are thought to originate from the clonal proliferation of bone marrow progenitors altered by somatic mutation.1 Erythrocytes from patients with paroxysmal nocturnal hemoglobinuria have been shown to be deficient in membrane glycoproteins, such as decay-accelerating factor (DAF)2 , 3 and a factor called either homologous restriction factor4 (HRF) or C8-binding protein5 (C8bp). DAF can inhibit the formation and induce the dissociation of C3 convertases of the classical and alternative pathways of complement activation.6 7 8 HRF/C8bp appears to limit the . . .